Factors that stimulate formation of new adipocytes during development of obesity are yet to be identified. We examined whether diet acts directly on preadipocytes to stimulate replication and differentiation or indirectly by interacting with adipocytes to release or modify local growth factors. Male Sprague-Dawley rats were fed chow or diets high in starch (HST), saturated (HFS) or polyunsaturated (HFP) fats until 5-7 months of age. We found that, compared to other diets, HFS induced acceleration of replication of preadipocytes in primary culture (doubling time of retroperitoneal-derived preadipocytes: HFS 17 +/- 1 versus chow 32 +/- 6 and HFP 29 +/- 3 h, P < 0.05). HFS stimulated greater expansion of retroperitoneal fat than HFP even when caloric intake was equal and increased adipocyte number threefold. Preadipocyte pool size in inguinal and retroperitoneal fat pads changed relative to fat pad weight in rats fed all diets compared to chow, suggesting that the balance between the number of cells capable of replicating and those terminally differentiated was perturbed. Differentiation of preadipocytes and release of adipocyte growth factors in vitro were unaffected by diet. We concluded that dietary saturated fats induced expansion of adipose tissue mass more effectively than polyunsaturated fats and that this may, in part, be achieved by acceleration of preadipocyte replication.