Osteoblasts synthesize and mineralize bone matrix and are principal target cells for parathyroid hormone (PTH). The type 1 PTH/PTH-related protein (PTHrP) receptor (PTH1R), cloned from rat osteoblastic cells, activates multiple intracellular signaling mechanisms. The specific roles of these PTH1R signals, or of responses to other types of PTH receptors that may be expressed, in regulating osteoblast function are incompletely understood. Use of established mammalian osteoblastic cell lines has led to much understanding of PTH action in bone, although such cells are of neoplastic origin or have other characteristics that compromise their validity as models of normal osteoblasts. To examine the role of the PTH1R in osteoblast biology, we have isolated a series of clonal murine calvarial osteoblastic cell lines that are only conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and that lack both functional alleles of the PTH1R gene. When cultured under nontransforming conditions, these cells stopped proliferating, expressed a series of characteristic osteoblastic genes (including the nonfunctional remnant of the PTH1R gene), and, after 3-4 weeks, produced mineralized bone nodules in a manner that was regulated by 1,25-dihydroxyvitamin D3 but not by PTH(1-84). Cyclic AMP measurements revealed no evidence of expression of alternate species of Gs-linked PTH receptors. Stable transfection with PTH1R cDNA reconstituted both PTH binding and adenylyl cyclase activation, increased basal osteocalcin expression, and supported PTH stimulation of c-Fos expression and matrix mineralization. These conditionally transformed, PTH1R(-/-) clonal osteoblastic cell lines should prove useful for studies of the regulation of osteoblast differentiation and function by both endogenous nonclassical species of PTH (or PTHrP) receptors and mutant signal-selective PTH1Rs.