Accumulation of [3H]L-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na(+)-dependent and Na(+)-independent components. The accumulation of [3H]L-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [3H]L-proline, [3H]L-glutamate and [3H]GABA. It was fully inhibited by L-glutamine, L-asparagine, L-cysteine, L-alanine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine, L-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by beta-alanine, taurine, glycine nor was it inhibited by the marker for the A system, L-2-methylamino isobutyric acid. D-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [3H]L-serine. We conclude that L-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na(+)-dependent one of lower affinity (K(m) in mM range) and a Na(+)-independent system of higher affinity (K(m) approximately 20-100 micro M). Characteristics of [3H]L-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical 'A', nor by the 'large neutral', amino acid transport systems but predominantly by transporters belonging to the recently identified LAT (L-amino acid transporter) family.