Prostate secretory epithelial cells have the unique and highly specialized function of accumulating and secreting extraordinarily high levels of citrate. Aspartate is a major four-carbon source of oxaloacetate required for the net synthesis of citrate by these cells. The prostate epithelial cells contain a Na(+)-coupled, high-affinity aspartate transporter which permits the cells to accumulate aspartate from circulation against a large concentration gradient. Because of this unique relationship between aspartate and citrate, it was important to determine if citrate modulated the high-affinity transport of aspartate into prostate epithelial cells. The current studies with freshly prepared prostate epithelial cells obtained from rat ventral prostate demonstrated that citrate exerted two effects on aspartate transport. Physiological levels of extracellular citrate (i.e., equivalent to circulating levels) markedly inhibited (cis-inhibitory effect) aspartate transport. In contrast, intracellular citrate at concentrations associated with normal in situ cells resulted in two levels of stimulation of aspartate transport. A 4-fold increase in aspartate transport occurred when the intracellular citrate was increased up to 500 nmols/g, and an 11-fold increase resulted when the intracellular citrate concentration was elevated to 1800 nmols/g (which is uniquely characteristic of prostate cells). The combined effect of the cis-inhibitory and trans-stimulatory actions of citrate was a consistent 1-2 fold increase in aspartate transport. These studies support the concept that citrate is a physiological regulator of the high-affinity transport of aspartate into prostate secretory epithelial cells in association with the unique and highly specialized function of net citrate production and secretion.