Although FSH up-regulates follicular cell X-linked inhibitor of apoptosis protein (XIAP) expression and suppresses apoptosis in vivo, if these events are coincidental or causally related remains to be investigated. The present study examined the role and gonadotrophic regulation of XIAP expression during follicular development in vitro. Follicles (160–210 μm) cultured for 0–6 days with FSH (100 ng/ml) showed significant growth, as evidenced by increases in follicular size, cell number, and DNA contents. Follicular XIAP content was low in the absence of FSH but was increased by the addition of gonadotropin. Apoptosis was evident in follicles cultured without FSH but was suppressed in the presence of gonadotropin. At low FSH concentration (5 ng/ml), adenoviral XIAP sense cDNA expression increased XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth. Infection of the FSH-stimulated follicles with XIAP antisense elicited opposite responses. In primary granulosa cell cultures, FSH significantly increased XIAP content, inhibited apoptosis, and decreased cell number, a response potentiated by XIAP sense expression. In conclusion, the present studies demonstrated, to our knowledge for the first time, that XIAP plays an important role in the regulation of ovarian follicular development. In addition, a follicle culture system coupled to an adenoviral gene-manipulation procedure has been established and may prove to be a useful approach in assessing the role of specific genes in follicular development and atresia.