Up to now the regulatory mechanisms, which govern the concentrations of neurosteroids in the brain, are unclear. Seasonal breeders may serve as excellent models to understand physiological changes in neurosteroid levels and their regulatory mechanisms. The present study first investigated immunohistochemically the localization of cytochrome p450 side-chain cleavage enzyme (p450scc) and 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(4)-isomerase (3betaHSD) in the brain of the newt Cynops pyrrhogaster, a seasonally breeding amphibian. Both p450scc- and 3betaHSD-like immunoreactive cells were restricted to the preoptic area. Seasonal changes in neurosteroid concentrations were then examined using adult males. Pregnenolone concentrations in the brain showed marked changes during annual breeding cycle and a maximal level in August, independent of the plasma steroid levels which were all low throughout the year. Progesterone concentrations in the brain, which were lower than pregnenolone levels, also showed peaks in April and August. In contrast, the pregnenolone sulfate level was low and its change was less pronounced. To clarify environmental factors that induce seasonal changes in neurosteroid levels, adult males were further subjected to different photoperiods and ambient temperatures for 5 weeks. Both pregnenolone and progesterone concentrations in the brain were significantly higher in the long day (LD) group than in the short day (SD) group, whereas no significant effects of different ambient temperatures on neurosteroid levels were detected. These results suggest that the newt brain possesses steroidogenic enzymes p450scc and 3betaHSD and exhibits seasonal changes in the pregnenolone and progesterone concentrations. Photoperiod may be a more important environmental factor than temperature for the regulation of neurosteroid levels in the brain.