High levels of immunoreactive and biologically active parathyroid hormone-related peptide (PTHrP) were detected in synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The levels of PTHrP immunoreactivity in synovial fluids, measured by a two-site immunoradiometric assay (IRMA) which detects hPTHrP(1-72) or longer peptides and a radioimmunoassay (RIA) specific to the carboxy-terminal portion of hPTHrP, were 3.2 +/- 0.3 pmol of hPTHrP(1-86)/l and 61 +/- 7.0 pmol of hPTHrP(109-141)/l in OA patients (mean +/- SE, n = 23), and 4.8 +/- 0.8 pmol of hPTHrP(1-86)/l and 164 +/- 30 pmol of hPTHrP(109-141)/l in RA patients (n = 26). Synovial fluid PTHrP levels distributed above the normal plasma reference ranges in each assay (0.7-2.6 pmol of hPTHrP(1-86)/l; 16-60.6 pmol of hPTHrP(109-141)/l). After concentration using sequential cation-exchange and reverse-phase chromatography, synovial fluid exhibited the activity that stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblastic ROS 17/2.8 cells expressing PTH/PTHrP receptors. The cAMP accumulation activity in synovial fluid was sensitive to coincubation with excess hPTHrP(3-40), a PTH/PTHrP receptor antagonist, and was completely neutralized by preincubation with a monoclonal antibody specific to hPTHrP but not PTH. Immunohistochemical analysis of RA synovium revealed that PTHrP was localized in fibroblast-like cells in the synovial pannus invading articular cartilage. Our data show that PTHrP is produced locally by the diseased synovial tissue and released into synovial fluid at high concentrations, allowing us to hypothesize that PTHrP plays a novel role as a paracrine/autocrine factor in the pathology of OA and RA.