Steroid synthesis in the human ovary is regulated by the temporal and spatial expression of enzymes involved in each step of ovarian steroidogenesis. Recent studies have demonstrated that DAX-1 and COUP-TFII negatively regulate adrenal 4 binding protein (Ad4BP)/steroidogenic factor-1 (SF-1)-dependent transcription of steroidogenic enzymes in experimental animals. In this study the expression patterns of these proteins in human normal ovaries were examined using immunohistochemistry and compared with those of steroidogenic enzymes. In the ovarian follicle and corpora lutea, immunoreactive DAX-1 protein was detected predominantly in granulosa cells, whereas COUP-TFII was identified in thecal cells. In granulosa cells, both immunoreactive DAX-1 and Ad4BP/SF-1 protein expression increased after the preantral follicular stage. DAX-1 immunoreactivity was relatively high compared with that of Ad4BP/SF-1 in all follicular stages from premordial to nondominant. The increase in expression of Ad4BP/SF-1 immunoreactivity between follicles in the preantral and dominant stages of follicular development was greater than that in DAX-1 expression. In thecal cells, immunoreactive COUP-TFII was consistently elevated throughout the menstrual cycle, whereas Ad4BP/SF-1 immunoreactivity increased according to the development of the follicle. These results indicated that DAX-1 and COUP-TFII may play a role in the modulation of Ad4BP/SF-1-dependent transcription of steroidogenic enzymes in different cell types and follicular stages in normal cycling human ovaries.