It is generally accepted that the phospholipid and calcium-dependent enzyme protein kinase C (PKC) plays a significant role in secretion of hormones from anterior pituitary cells. The present study was undertaken to study age and sex-related changes in 1. levels of immunoreactivity of PKC isozymes and 2. distribution of immunoreactivity of PKC isozymes after stimulation with substance P (SP) in rat lactotroph-enriched cell cultures. The alpha, beta, delta and zeta isozymes were present in both sexes and at all ages. There was a sex-specific differential regulation of the different PKC isozymes as a function of sexual maturation. In male rats there was an up-regulation of the alpha isozyme throughout the sexual development, while the beta subtype showed a small, but significant decrease in immunoreactivity with increasing age. In female rats, on the other hand, the beta species was up-regulated with increasing age while the other subtypes remained constant. The concentration of the delta and zeta isozymes was unaffected of sex and age. Stimulation of lactotroph-enriched cell cultures with substance P (SP) resulted in translocation of the alpha and beta isozymes from the soluble to the particulate fraction while the delta and zeta species were left unchanged independently of age and sex. However, a decrease in responsiveness was observed in adult male rats, although a significant degree of translocation of alpha and beta species was still detected. On the basis of these results it is suggested that in lactotroph-enriched cell cultures basal levels of PKC subtype immunoreactivity and distribution of immunoreactivity of PKC isozymes after SP challenge might be regulated as a function of sex and age.