Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin receptor (CTR) to also function as a high-affinity amylin receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTR(I1-)) or the insert positive (hCTR(I1+)), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for amylin binding. Also, in CHO-P, hCTR(I1-) induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTR(I1+) induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/RAMP-generated receptor, whereas in CHO-P cells a two-affinity state receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate receptor phenotype.