To examine the autocrine/paracrine effect of parathyroid hormone-related peptide (PTHrP) on osteoblast function, the entire coding region of rat PTHrP (1-141) cDNA inserted into the expression vector was stably transfected into the rat clonal strain of the osteoblast-like cell, ROS 17/2.8, and established stable transfectants. Using the PTHrP-overexpressing ROS cells (ROS/PLP/6), we analyzed in vitro cell characterization and in vivo osteogenic properties. As expected, overexpression of endogenous PTHrP in vitro induced PTH/PTHrP receptor down-regulation confirmed by Northern blots, receptor binding assays, and functional analysis. The established transfectants indicated a decreased growth rate compared with the original non-transfected ROS 17/2.8. Although cAMP production induced by exogenous PTH was suppressed in ROS/PLP/6, the stimulatory effects of forskolin and chorela toxin showed no significant difference between the original ROS 17/2.8 and transfected cells, but the in vivo osteogenic properties were histologically potentiated in transfectants with increased bone matrix and acceleration of mineralization within tumors. The levels of osteocalcin and osteopontin mRNAs were also increased in transfectants. The down-regulated in vitro PTH/PTHrP receptor mRNA was restored in in vivo tumor tissues. Our study provides clear evidence that the in vivo osteogenic function in ROS cells is potentiated by PTHrP, through an autocrine/paracrine mode of action.