Parathyroid hormone-related peptide (PTHrP) may be synthesized in or near its target tissues and acts by autocrine and/or paracrine fashions. The rat clonal strain of the osteoblast-like cell, ROS 17/2.8-5, can express low levels of PTHrP in the cytoplasm; however, the autocrine function of PTHrP in ROS cells has not yet been clarified. We created PTHrP expression vectors and transfected them into cells in order to elucidate the functional role of PTHrP on their own target cells. Sense and antisense rat PTHrP expression vectors (pSV2 neo-ECE-rPLP) were transfected into ROS cells independently and cultured for 72 hours. Cells overexpressing PTHrP were detected by immunocytochemical analysis and confirmed by Northern blot analysis, respectively. These transfected cells demonstrated mitogenic activity as determined by BrdU uptake staining. These findings suggest the functional role of PTHrP on their own PTHrP expressing cells via an autocrine/paracrine fashion. These PTHrP-overexpressing ROS cells provide a model in vitro system to clarify the mechanism by which PTHrP acts in an autocrine/paracrine fashion.