The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys 2 7 in myc-tagged human (h) RAMP1 was deleted (hRAMP1Δ1), and Cys 4 0 , Cys 5 7 , Cys 7 2 , Cys 8 2 and Cys 1 0 4 were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Δ1 or -C82A, cell surface expression, [ 1 2 5 I]hαCGRP binding and cAMP formation in response to hαCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [ 1 2 5 I]hαCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by hαCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC 5 0 . But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys 2 7 or the substitution of Cys 8 2 by Ala in hRAMP1, but it was impaired by the substitution of Cys 4 0 , Cys 5 7 , Cys 7 2 and Cys 1 0 4 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.