The isoforms of SREBP (-1a, -1c and -2) are key transcriptional regulators of lipid biosynthesis. We examined their regulation by gonadotrophin and insulin in human granulosa cells. Following removal of leukocytes, granulosa cells were exposed to hormonal additions for 16 h starting on day 2 of culture. Progesterone, lactate and IGFBP-1 were measured in culture medium and cellular mRNA measured by competitive RT-PCR. Addition of hCG (100 ng/ml) stimulated progesterone production (7.0-fold, P < 0.001 vs. control), while lactate was increased by hCG (1.6-fold, P < 0.001) and insulin (1.4-fold, P < 0.001; 1000ng/ml). Insulin decreased IGFBP-1 production by 85% (P < 0.001). There were no significant effects on the expression of SREBP-1a, but significant increases in mRNA for SREBP-1c with insulin (6.3-fold), hCG (10.4-fold) and in combination (15.2-fold; P < 0.01 for all comparisons). No consistent effects on SREBP-2 were observed. The expression of mRNA for fatty acid synthase, a target gene for SREBP-1c, was increased by hCG (24-fold, P = 0.006) and insulin (19-fold, P = 0.024), which also increased the level of cellular, total fatty acid (1.34-fold. P = 0.03). Thus hCG and insulin cause a switch toward expression of the SREBP-1c isoform with consequent effects on fatty acid synthesis. We suggest that high circulating insulin, associated with clinically-defined insulin resistance, may up-regulate SREBP-1c expression in the ovary.