To examine the essential mechanisms of steroid production in ovarian theca cells, we analyzed the expression of genes associated with steroid production using simple culture system with serum medium. In addition, we examined the involvement of DAX-1, COUP-TFII, and Ad4BP/SF-1 transcription factors on the steroid production in theca cells. Theca cells begin to display an elongated or fibroblastic aspect within 24 h of culture. Over the next 48 h, they metamorphosed from the fibroblastic to the epitheloid phenotype. The number of theca cells increased during culture period. Androstenedione and progesterone production per cell decreased at 48-96 h compared with 0-48 h of culture. Steroidogenic acute regulatory protein (StAR) and CYP 17 genes expression decreased at 48 h compared with 0 h of culture, and afterward maintained a low level. In contrast, expression of 3beta-HSD and P450scc mRNAs increased at 48 h compared with 0 h of culture. Protein expression of Ab4BP/SF-1 maintained a constant level during culture. COUP-TFII protein expression showed a peak level at 24 h of culture period. DAX-1 protein expression began to increase at 48 h of culture. Our data suggested that the inhibition in CYP 17 and StAR genes by DAX-1 transcription factor may be associated with the decrease in androstenedione and progesterone production by theca cells during in vitro culture. Such an essential pathway for steroid production might indicate the importance of theca cell function in bovine ovary.