Our laboratory has recently cloned and expressed a brain- and neuron-specific Na(+)-dependent inorganic phosphate (P(i)) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na(+)-dependent 32Pi cotransport in cultured fetal rat cortical neurons, where > 90% of saturable P(i) uptake is Na(+)-dependent. Saturable, Na(+)-dependent 32Pi uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na(+)-dependent P(i) transport was optimal at physiological temperature (37 degrees C) and pH (7.0-7.5), with apparent Km values for P(i) and Na+ cf 54 +/- 12.7 microM and 35 +/- 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (> 60%) Na(+)-dependent P(i) uptake, with a threshold for maximal P(i) import of 1-2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na(+)-dependent P(i) import rates. These data establish the presence of a Na(+)-dependent P(i) cotransport system in neurons of the CNS, demonstrate the Ca(2+)-dependent nature of 32Pi uptake, and suggest that the neuronal Na(+)-dependent P(i) cotransporter may import P(i) required for the production of high-energy compounds vital to neuronal metabolism.