The basic pathway of oocyte development and its regulation is evolutionarily conserved among vertebrates; however, little is known about the role of hormones at the first stages (Stages I and II) of follicle development in fish. In the present study, zebrafish follicles at Stages I and II were exposed in vitro to the reproductive hormones 17β-oestradiol (E2), 11-ketotestosterone (11KT), 17,20β-dihydroxy-4-pregnen-3-one (DHP) and to the secondary messenger dibutyryl cyclic adenosine monophosphate (db-cAMP) at a concentration of 1µM for a 48-h period. Morphological alterations of the ooplasm were assessed by transmission electron microscopy and of the granulosa cell layer by quantitative stereology. Expression of mRNA was analysed for cell-cycle genes (cyclin B and E) and resident proteins of the endoplasmic reticulum (calnexin and 78-kDa glucose-regulated protein (grp78/bip)). E2 and db-cAMP stimulated the presence of endoplasmic reticulum in the ooplasm and calnexin mRNA increased in the db-cAMP treatment, but also in response to 11KT and DHP. 11KT, DHP and db-cAMP inhibited the progression of the cell cycle in the granulosa-theca cell layer, indicated by a reduction of the nucleus volume-weighted size of granulosa cells and of increased cyclin E mRNA expression. Reproductive hormones had different effects on the ooplasm and the granulosa-theca cell layer of zebrafish follicles, predominantly at Stage II.