Intrasequence comparison of the type IIa Na(+)-P(i) cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. Mutants N199C and V202C were fully inhibited after methanethiosulfonate ethylammonium exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant methanethiosulfonate ethyltrimethylammonium, which suggested a sidedness of Cys modification. Compared with the wild-type (WT), mutant A203C showed altered Na(+) leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferring substrate affinity, other mutations at this site were made. Only two mutants yielded significant (32)P(i) uptake and inward P(i)-induced currents with decreased P(i) affinity; for the others, P(i) application suppressed only the Na(+) leak. We suggest that ICL-1 and ECL-3 sites contribute to the transport pathway and that site N199 is implicated in defining the transport mode.