N-Terminal analogs of PTH-related protein (PTHrP) and PTH bind to a common receptor and exhibit similar biological properties. However, recent studies suggest that certain midregion and C-terminal PTHrP peptides have activities distinct from those of PTH in the placenta and in osteoclasts, respectively. In this study we determined the biological activities of full-length recombinant PTHrP-(1-141) and several synthetic N-terminal, midregion, and C-terminal PTHrP fragments in two PTHrP-producing cell types. Peptides were tested for their ability to stimulate cAMP production and raise intracellular free calcium ([Ca2+]i) in primary rat aortic smooth muscle cells (VSMC) and UMR-106 rat osteoblast-like (UMR) cells. In UMR cells PTHrP-(1-34)NH2, PTHrP-(1-141), and bovine PTH-(1-34) all increased cAMP (approximately 50 fold) and [Ca2+]i (180 nM). By contrast, in VSMC, these N-terminal peptides increased cAMP (3-fold) but had no detectable effect on [Ca2+]i. PTHrP-(1-34) and PTHrP-(1-141) significantly blunted the angiotensin II-induced rise in cAMP (but not the calcium signal) consistent with the concept that PTHrP opposes angiotensin II activity in VSMC. PTHrP-(67-86)NH2, PTHrP-(107-138)NH2, and PTHrP-(107-111)NH2 had no effect on either cAMP or [Ca2+]i in either cell type. VSMC and UMR-106 cells both expressed a 2.5-kilobase PTH/PTHrP receptor messenger RNA (mRNA) transcript. However, high affinity specific binding of 125I-labeled [Tyr36] PTHrP-(1-36)NH2 was detected in UMR cells but not in VSMC. We conclude that the PTH-like, N terminus of the PTHrP molecule is critical in induction of cAMP and [Ca2+]i pathways in UMR cells, and for cAMP stimulation in VSMC. In addition, PTHrP, like other established vasodilators, signals in VSMC mainly (if not exclusively) by increasing the production of cAMP.