This study explored several physiological criteria that could be used to assess the steroidogenic condition of the ovarian follicles of individual fish of an asynchronously ovulating captive rainbow trout Oncorhynchus mykiss stock. In these fish, the date of sampling, morphological variables such as gonado-somatic index or ovarian follicle mass and visual assessment of the ovary provided accurate indications of the maturational condition of an individual. The physiological variables measured included the in vitro basal and cyclic adenosine monophosphate (cAMP)-stimulated synthesis by ovarian follicles of 17?-oestradiol (E(2)) and testosterone (T); in addition, quantitative reverse-transcription (RT)-PCR was used to measure the relative expression of star and p450scc genes by ovarian follicles. The ratios of cAMP-stimulated E(2) and T synthesis to basal E(2) and T synthesis provided a reliable indication of differences in the steroidogenic status of the follicles of individual animals. On the basis of these criteria, together with the use of gene expression profiles, it was possible to classify individual fish as being at an early, mid or late-vitellogenic stage.