Bone marrow contains osteoblast progenitor cells that can be obtained with aspiration and appear to arise from a population of pluripotential connective-tissue stem cells. When cultured in vitro under conditions that promote an osteoblastic phenotype, osteoblast progenitor cells proliferate to form colonies of cells that express alkaline phosphatase and, subsequently, a mature osteoblastic phenotype.We evaluated the number of nucleated cells in bone-marrow samples obtained with aspiration from the anterior iliac crest of thirty-two patients without systemic disease. There were nineteen male patients and thirteen female patients; the mean age was forty-one years (range, fourteen to seventy-seven years). The prevalence and concentration of the osteoblast progenitor cells also were determined, by placing the bone-marrow-derived cells into tissue-culture medium and counting the number of alkaline phosphatase-positive colony-forming units. In order to assess the effect of aspiration volume, two sequential experiments were performed. In the first experiment, aspiration volumes of one and two milliliters were compared. In the second experiment, aspiration volumes of two and four milliliters were compared.The mean prevalence of alkaline phosphatase-positive colony-forming units in the bone-marrow samples was thirty-six per one million nucleated cells (95 per cent confidence interval, 28 to 47); a mean of 2400 alkaline phosphatase-positive colony-forming units was obtained from a two-milliliter aspirate. There was a significant difference among the patients with respect to the number of alkaline phosphatase-positive colony-forming units in these bone-marrow samples (p < 0.001). Seventy per cent of this variation in the prevalence was due to variation among patients, and 20 per cent was due to variation among aspirates.The number of alkaline phosphatase-positive colony-forming units in the aspirate increased as the aspiration volume increased. However, contamination by peripheral blood also increased as the aspiration volume increased. An increase in the aspiration volume from one to four milliliters caused a decrease of approximately 50 per cent in the final concentration of alkaline phosphatase-positive colony-forming units in an average sample.CLINICAL RELEVANCE: On the basis of these data, we recommend that, when bone marrow is obtained with aspiration for use as a bone graft, the volume of aspiration from any one site should not be greater than two milliliters. A larger volume decreases the concentration of osteoblast progenitor cells because of dilution of the bone-marrow sample with peripheral blood. We estimate that four one-milliliter aspirates will provide almost twice the number of alkaline phosphatase-positive colony-forming units as will one four-milliliter aspirate. In addition, these data confirm that humans differ significantly from one another with respect to the cellularity of bone marrow and the prevalence of osteoblast progenitor cells. Additional studies are necessary to determine if the number or prevalence of alkaline phosphatase-positive colony-forming units in bone marrow is a determining factor in the efficacy of an autogenous bone or bone-marrow graft and to ascertain how the number and function of alkaline phosphatase-positive colony-forming units may change as a function of factors such as age, menopausal status, and selected diseases.