The prostate gland produces and secretes extraordinarily high levels of citrate. Studies with rat ventral prostate (VP) have demonstrated that aspartate can serve as a four-carbon source of oxalacetate in the synthesis of citrate. To achieve this, prostate secretory epithelial cells must contain a transport system for the active uptake of aspartate from circulation. The present studies with VP epithelial cells confirm the existence of a Na(+)-dependent high-affinity L-aspartate transporter. The transporter has an optimal pH approximately 7.5 and is temperature dependent. It appears to be an anionic amino acid transporter capable of transporting L-glutamate but not basic or neutral amino acids. The transporter is inhibited by ATPase inhibitors, thereby indicating its dependency on a Na+ gradient. The characteristics of the high-affinity L-aspartate transporter are consistent with its operation at the basilar membrane for the transport of circulating aspartate into the cell. Castration (24 hr) resulted in a significant decrease in the ability of VP epithelial cells to transport L-aspartate. The administration of testosterone to castrated rats completely restored L-aspartate transport. In addition, in vitro testosterone addition (10(-8) M for 30 min) to isolated prostate epithelial cells markedly increased L-aspartate transport. Both cycloheximide and actinomycin inhibited the testosterone effect. The studies reveal that testosterone is a regulator of this Na(+)-dependent high-affinity L-aspartate transporter. The mechanism of this testosterone effect appears to involve both RNA and protein synthesis. We now have a model system to elucidate this novel effect of testosterone.