Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and gamma-aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1-day-old rats. The Na(+)-dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system Nb. System Nb activity can be monitored by assaying Na(+)-dependent [3H]glutamine uptake in the presence of 2 mM concentrations of both 2-(methylamino) isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system Nb for glutamine, as estimated from K(m) values, is significantly greater than that observed for the hepatic and muscle Na(+)-dependent glutamine transporters, systems N and Nm. In sharp contrast to the hepatic system N transporter, system Nb exhibits a relative insensitivity to pH and does not permit Li+ substitution for Na+ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.