The objective of these experiments was to develop an in vitro cell culture system for differentiation of bovine preadipocytes, which will permit examination of differences in differentiation between intramuscular (i.m.) and subcutaneous (s.c.) bovine preadipocytes. Stromal-vascular cells from bovine i.m. and s.c. adipose depots were isolated and cultured. Clonally derived s.c. preadipocytes were used to determine the ability of insulin, bovine serum lipids, octanoate, acetic acid, dexamethasone (DEX), and troglitazone (TRO) to elicit differentiation of these cells when added to serum-free medium. Addition of 10 and 20 microL/mL of a commercially available serum lipids supplement to low-glucose Dulbecco's modified Eagle's medium containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity (P < 0.01). Inclusion of 1.25 to 10 microM TRO to medium containing 280 nM insulin and 20 microL/ mL serum lipids supplement also increased GPDH activity (P < 0.001) compared with 0 microM TRO. The combination of 280 nM insulin, 1 mM octanoate, and 10 mM acetic acid, with 48 h exposure to 0.25 microM DEX caused morphological differentiation in a small number of cells but did not stimulate GPDH activity (P = 0.99). When used together, 280 nM insulin, 20 microL/mL of serum lipids supplement, 40 microM TRO, and 0.25 microM DEX stimulated differentiation compared with the aforementioned treatment (P < 0.001). Omission of TRO or insulin from this medium reduced GPDH activity by 68% (P < 0.001), whereas removal of DEX tended to reduce GPDH activity (P = 0.06). Preadipocytes from s.c. (n = 3) and i.m. (n = 2) adipose tissues of 3 steers were used to determine the effects of TRO on differentiation using the established conditions. Forty to sixty microM TRO enhanced differentiation compared with 0 microM TRO (P < 0.02) in both depots. No depot differences in response to TRO were detected (P = 0.32). These data demonstrate that bovine preadipocytes are capable of differentiation in response to combinations of insulin, serum lipids, DEX, and TRO. Although TRO enhanced differentiation of bovine preadipocytes, no differential effects of TRO on the differentiation of s.c. and i.m. cells were detected.