The aim of this study was to investigate the role of cell replication for the differentiation of human adipocyte precursor cells in primary culture. When cells were seeded in a medium supplemented with 10% fetal bovine serum, they started to proliferate within 48 h after exposure, as assessed by cell counting and [3H]thymidine autoradiography. When cells were inoculated in the absence of serum, a significant degree of cell proliferation was not detectable. Histochemical investigations using bromodeoxyuridine incorporation demonstrated that cells replicating their DNA did not accumulate lipid droplets. Inoculating adipocyte precursor cells under completely serum-free conditions resulted in a 30-50% higher expression of lipogenic enzymes such as glycerol-3-phosphate dehydrogenase and lipoprotein lipase than keeping cells in serum-supplemented medium for the initial 16 h. Addition of cytosine arabinoside at concentrations that effectively block mitosis did not interfere with adipocyte development. In conclusion, adipocyte precursor cells from human adipose tissue do not require cell division to enter the differentiation process in vitro. These cells may have already undergone possibly critical cell divisions in vivo and may be in a late stage of adipocyte development.