Conjugated linoleic acids (CLAs) reduce fat deposition in several mammalian species. Among the proposed mechanisms for this effect are reduced preadipocyte proliferation and differentiation. We measured proliferation and differentiation of cultured human preadipocytes treated with CLAs. Preadipocytes were differentiated with insulin, hydrocortisone, transferrin, and 10% fetal bovine serum, with isobutyl-methylxanthine included for the first 2 d. The differentiation medium contained 200 μM oleic acid (C18:1), 50 μM cis-9,trans-11-CLA (9,11-CLA), or 50 μM trans-10,cis-12-CLA (10,12-CLA); the negative control medium contained no added fatty acid, and the cells did not differentiate. Cell number increased three to four times during the 17 d of differentiation, but was 30–35% lower in the CLA-treated cells than in the negative control cells. Compared with the negative control cells, differentiation was increased in the cells treated with C18:1 (increased Oil Red O–stained material [OROSM], triacylglycerol, glycerol 3-phosphate dehydrogenase activity [GPDH], peroxisome proliferator–activated receptor-γ [PPARγ] messenger ribonucleic acid [mRNA], and lipoprotein lipase [LPL] mRNA). In effect, the C18:1–treated cells act as a positive control to demonstrate the differentiation capacity of each cell lot. Both 9,11-CLA– and 10,12-CLA–treated cells had increased differentiation (increased OROSM, triacylglycerol, GPDH, PPARγ, and LPL) compared with the negative control cells. The data suggest that early in differentiation when de novo fatty acid (FA) synthesis is limited and competition for FAs by membrane and triacylglycerol synthetic pathways is great, human preadipocytes do not differentiate unless a PPARγ ligand is added. Either CLA isomer or C18:1 can provide such a ligand.