The human teratocarcinoma cell line NTera2/D1 can be differentiated to produce post-mitotic neurones (NT2-N cells) by prolonged (> 3 week) exposure to retinoic acid. In this study, we describe the characterisation of high-affinity Na+-dependent L-glutamate transport activity in post-mitotic differentiated NT2-N cells. NT2-N cells, but not the undifferentiated precursor cells, transported L-glutamate in a Na+-dependent manner, as determined by equimolar replacement of Na+ with choline. L-glutamate uptake was saturable and Eadie-Hofstee transformation of the saturation data revealed a Km of 10.6+/-0.8 microM, and a maximum transport capacity (Vmax) of 100.3+/-12.3 pmol min(-1) mg(-1) protein. Pharmacological characterisation of the transport activity in NT2-N cells produced a rank order of inhibitory activity which was identical to that determined for the human excitatory amino acid transporter 2 which we have analysed in a stable mammalian cell line (Madin Darby Canine Kidney (MDCK) cells). Of particular note, L-glutamate transport by NT2-N cells was sensitive to both dihydrokainate and kainate. The expression of human excitatory amino acid transporter mRNAs was studied using reverse transcriptase polymerase chain reaction. NT2-N cells expressed transcripts for excitatory amino acid transporters 2 and 3, but not for the subtypes 1, 4 and 5. We conclude that although the mRNA expression studies suggest the presence of transcripts for both excitatory amino acid transporter 2 and 3 in NT2-N cells, the sensitivity to dihydrokainate and kainate determined in the pharmacological analysis indicates that, of the known transporter subtypes, excitatory amino acid transporter 2 contributes to the bulk of the L-glutamate transport activity present in these cells.