We attempted to express point-mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point-mutant receptors (C24→S, C44→S, C53→S, C67→S, C85→S and C101→S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point-mutant receptors (C11→S, C186→S, C193→S and C263→S) were able to bind poorly 125I-secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263→S), the four triple mutants, and through double mutants C186,193→S and Cl86,263→S with a very high (μM) EC30 value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11–C186, and between C193–C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11–C186 (but not the C193–C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.