Treatment of rat renal brush-border membrane vesicles with N,N'-dicyclohexylcarbodiimide (DCCD) causes irreversible inhibition of the Na+-coupled transport systems for D-glucose, L-phenylalanine, L-glutamate, and sulfate. The DCCD-reactive side groups of these transport systems differ in their sensitivity towards DCCD and protection by substrates. The D-glucose and L-glutamate transporters cannot be protected by their substrates. In contrast, Na+ protects the transport systems for L-phenylalanine and sulfate from inactivation by DCCD. The data suggest covalent modification by DCCD of D-glucose and L-glutamate transporters apart from their substrate binding sites and of L-phenylalanine and sulfate transporters within their Na+-binding regions.