To understand better the mechanism by which 5-alpha-dihydrotestosterone (5-alpha-DHT) influences prostate epithelial cell function, we examined the effects of 5-alpha-DHT on phosphoinositide metabolism in human prostate cancer cell lines. Androgen receptor-positive LN-CaP cells showed dose-responsive, steady-state elevations in phosphoinositide metabolism when treated with 5-alpha-DHT. The intracellular pool of 3H-myoinositol decreased and the incorporation of 3H-myoinositol into cellular lipids increased with increasing concentrations of 5-alpha-DHT. 5-alpha-DHT increased the release of 3H-inositol phosphates into the media. The inactive stereoisomer, 5-beta-DHT, did not increase phosphoinositide metabolism. In androgen receptor-negative cells, phosphoinositide metabolism was not altered by 5-alpha-DHT. The slow induction of phosphoinositide metabolism by 5-alpha-DHT suggests that the effects may be mediated through other factors that serve as intermediates in 5-alpha-DHT modulation of intracellular signalling. We conclude that this modulation involves increased turnover of phosphatidylinositol, incorporation of myoinositol into cellular lipids, and alterations in the aqueous intracellular myoinositol pool size, possibly as a result of altered transport mechanisms.