Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRP-R) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRP-R expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classical subtype-1 receptor (CGRP-R₁). The aim of this study is to further characterize the MG63 CGRP-R using a selective agonist of the putative CGRP-R₂, [Cys(Acm)^2,7]-CGRP, and a relatively specific antagonist of CGRP-R₁, CGRP_8-37. [Cys(Acm)^2,7]-CGRP acted as a significant agonist only on ERK dephosphorylation, whereas this analogue effectively antagonized CGRP-induced cAMP production and phosphorylation of CREB and p38-MAPK. Although having no agonistic action when used alone, CGRP_8-37 potently blocked CGRP actions on cAMP, CREB, and p38-MAPK, but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA₂ value for ERK is clearly distinguishable from those of the other three plots, as judged by the 95% confidence intervals. Additional assays using isobutyl-methylxanthine or the PKA inhibitor, H-89, indicate that the cAMP-dependent pathway is predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38-MAPK phosphorylation. In consideration of previous data from Scatchard analysis of [¹²⁵I]-CGRP binding, these results suggest that MG63 cells possess two functionally distinct CGRP-R subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogues: one is best characterized as a variation of CGRP-R₁, and the second may be a novel variant of CGRP-R₂.