Uptake of L-[(14)C]citrulline was studied in cell culture models of the main neural cell populations, in astroglia-rich primary cultures derived from neonatal rat brain, in rat glioma cells C6-BU-1, in cells of the murine microglial clone N11 and in the glioma x neuroblastoma hybrid cell line 108CC15 with neuronal properties. For comparison, cells of the peripheral macrophage cell line RAW 264.7 were also investigated. A saturable component of uptake was found in all cases with K(M) values between 0.4 and 3.4 mM and V(max) values between 15 and 35 nmol.min(-1).(mg protein)(-1). A nonsaturable component dominated uptake at high concentrations of extracellular citrulline. Rates of uptake of L-citrulline were not affected when Na(+) or Cl(-) were omitted from the incubation medium or in the presence of depolarizing concentrations of K(+). Saturable uptake of citrulline was strongly inhibited by an excess of histidine or beta-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid; excess amounts of arginine, creatine, glutamate, cysteic acid or N-methyl-alpha-aminoisobutyric acid did not reduce citrulline uptake. Preincubation of the cells with bacterial lipopolysaccharide and interferon gamma did not stimulate transport of citrulline. The results suggest that at physiological concentrations citrulline is taken up by neural cells with the help of transport system L for large neutral amino acids. Therefore, in the brain, effective utilization of extracellular citrulline as part of an intercellular trafficking of intermediates of an NO/citrulline cycle depends on the concentrations of all neutral amino acids present.