We previously demonstrated that maternal protein restriction (MPR) during pregnancy and lactation led to fetal growth restriction and development of increased visceral adiposity in adult male rat offspring. Here we studied the rate of proliferation and differentiation of adipocyte precursors (preadipocytes) in vitro to investigate whether MPR may permanently program adipocyte growth and development in adult male offspring. Preadipocytes were isolated from visceral adipose tissue of control and MPR offspring at 130 days of age, and cultured under standard conditions. The rate of proliferation was studied by [(3)H]-thymidine incorporation, and the rate of differentiation assessed with the use of biochemical and morphological markers. Although it did not affect the rate of differentiation, MPR increased the rate of preadipocyte proliferation by almost twofold. To ascertain if the increased proliferation was due to persisting in vivo influences or aberrations inherent in the precursor cells, we studied the rate of preadipocyte proliferation in subcultures. We found that the increased rate of proliferation of MPR preadipocytes persisted throughout the first two subcultures, indicative of an inherent abnormality. In addition, we examined the rate of preadipocyte proliferation under reduced serum conditions. We showed that MPR reduced the rate of preadipocyte proliferation to 56 and 35% of the control in the presence of 5 and 2.5% serum, respectively. Taken together, these results demonstrate that MPR permanently programs adipocyte growth and development such that adipocyte precursors derived from MPR offspring replicate excessively under standard culture conditions but exhibit markedly attenuated growth rate under reduced serum conditions.