The transport of aspartate was measured in isolated prostate cells. Two kinetically distinct uptake systems of high (Km = 10.0 microM) and low (Km = 0.8 mM) affinity were observed. Transport by both systems was Na+ dependent and saturable. The tissue distribution of aspartate was also determined by measuring the washout kinetics of endogenous and radiolabeled aspartate from prostate fragments. The distribution of aspartate resulted in 47% of aspartate in the intracellular compartment and 52% in the extracellular (presumably) lumenal compartment. The distribution of aspartate resulted in a calculated intracellular to plasma concentration gradient of approximately 43. We concluded that prostate cells maintain a considerable intracellular to plasma gradient for aspartate and that these cells contain a transport mechanism capable of uptake of aspartate against this considerable gradient.