The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 microg/ml, transferrin 5 microg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.