A unique characteristic and function of rat ventral prostate, like human prostate, is the accumulation and secretion of high levels of citric acid. Aspartate is a proposed four-carbon precursor of citrate via transamination. Replenishment of endogenous aspartate requires continuous uptake of aspartate from circulation. This study was designed to identify aspartate transporters in isolated rat ventral prostate epithelial cells. The results indicated that two aspartate transporters, a high affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.01 mM) and a low affinity transporter (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.8 mM) exist in these cells. Both transporters are Na{dollar}\sp+{dollar}-dependent and pH sensitive. The optimal pH for the high affinity transporter is about 7.5, whereas for the low affinity transporter the optimum is between 6.5 and 7.0. The high affinity transporter is also temperature dependent. Competitive inhibitory studies indicate that L-aspartate uptake by the high affinity transporter is inhibited by L-glutamate and D-aspartate, but not by L-alanine and L-lysine. The low affinity system is inhibited by D-aspartate, but not by L-glutamate or L-alanine. These different characteristics suggest that the high affinity and the low affinity transporters are two distinct systems. The high affinity aspartate transporter is sensitive to the Na{dollar}\sp+{dollar}-K{dollar}\sp+{dollar} ATPase inhibitor vanadate but less sensitive to ouabain. This suggests that an ouabain-insensitive Na{dollar}\sp+{dollar}-ATPase exists on the cell membrane. High affinity aspartate uptake is not dependent on K{dollar}\sp+{dollar}. However, a Na{dollar}\sp+{dollar}-H{dollar}\sp+{dollar} antiport might be involved. Aspartate uptake is stimulated by testosterone in vivo and in vitro. The in vitro effect is rapid and is inhibited by cycloheximide and actinomycin D. Prolactin also stimulated aspartate uptake independent of testosterone and is inhibited by cycloheximide. The high affinity aspartate transporter is subject to transstimulation by aspartate and citrate.