Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentration of DTT (dithiothreitol) resulted in a decrease in the binding of [I-125]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [I-125]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha -melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha -MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [I-125]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha -MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha -MSH as well as NDP-MSH) generated a cAMP signal in response to alpha -MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at C78G mutant receptor. These findings demonstrate that (i) alpha -MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha -MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at mutant C78G receptor.