Disruption of the PTHrP gene has been shown to lead to abnormalities in growth plate resulting in abnormal bone development. Interestingly, the number of osteoclasts appears to increase in the metaphyses of PTHrP-deficient mice bone (J Cell Biol 126: 1611, 1994). We previously reported that PTHrP is produced by primary and several clonal osteoblastic cells in vitro. In primary osteoblastic cells, there was an inverse relationship between the amount of PTHrP mRNA expressed and the amount of PTH/PTHrP receptor mRNA (J Cell Physiol. in press, 1995). These findings suggest that PTHrP is physiologically important paracrine factor in bone. We developed a co-culture system of mouse hematopoietic and primary osteoblastic cells, with which to investigate osteoclast development in vitro. In this study, the role of PTHrP production by osteoblastic cells on osteoclast formation was examined using PTHrP-deficient mice. Calvaria, spleen, and tail were obtained from each fetus littermate (18.5 gestational) from pairs of heterozygous (+/-) mice. The phenotypes were determined by Southern blot analysis of tail genomic DNA using the specific primers for PTHrP. When spleen cells prepared from PTHrP (-/-) mice were co-cultured with normal (+/+) mice-derived osteoblastic cells for 7 days, many TRAP-positive multinucleated osteoclasts were formed in the presence of 1α,25(OH)2D3 (10 - 8 M) or PTHrP (1-34) (100 ng/ml). Similarly, the lack of PTHrP in osteoblastic cells were just as effective at generating mature osteoclasts from normal (+/+) mice-derived spleen cells in response to either 1α,25(OH)2D3 or PTHrP. Furthermore, PTHrP (107-139) (200 ng/ml) was ineffective at inhibiting osteoclast development in any of the co-cultures. These results indicate that the PTHrP production by osteoblastic cells does not mediate osteoclast differentiation in vitro.