Primary culture model of mature rat prostatic epithelial cells was established and the effect of prolactin (PRL) and dihydrotestosterone (DHT) on the secretory function was evaluated. Minced rat prostates were exposed to collagenase and the dispersed cells were filtered through a cell sieve. The cells were seeded in 5% FCS-supplemented McCoy's 5A medium for 16-20 hours, and the epithelial cells in suspension were transferred into plates and cultured in medium supplemented with 5% FCS, insulin, epidermal growth factor, cholera toxin and transferrin. This procedure resulted in enriched epithelial cells in culture. The plated cells were challenged with PRL and DHT for 48 hours, and the media and cells were collected for the determination of prostatic acid phosphatase (PAP) and DHT. The results showed that PRL stimulated production and secretion of PAP but did not synergize with DHT. PRL was invalid to affect DHT production in the prostatic epithelial cells. The present study indicated that the established culture model could be used to investigate hormonal effects on prostatic cells in vitro and PRL directly promoted the secretory function of the mature rat prostatic epithelial cells in culture.