L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.