The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.