INTRODUCTION Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration. METHODS The effect of conditioned serum on PG metabolism of cultured OA cartilage explants was compared to unconditioned serum. The effect of serum conditioning on levels of interleukin-1beta (IL-1beta), IL-4, IL-6, IL-10, IL-13, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG), oncostatin M (OSM), interleukin-1 receptor (IL-1ra) and transforming growth factor beta (TGF-beta) were measured by multiplex ELISA. As TNF-alpha levels were found to be increased in conditioned serum, the effect of TNF-alpha inhibition by etanercept on PG metabolism was studied in cartilage explants cultured in the presence of conditioned serum. Furthermore, cytokine levels in SF were measured three days after intraarticular ACS injection in OA patients to verify their retention time in the joint space. RESULTS PG metabolism was not different in the presence of conditioned serum compared to unconditioned serum. Levels of the anti-inflammatory cytokines IL-1ra, TGF-beta, IL-10 as well as of pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha and OSM were increased. IL-4, IL-13 and IFN-gamma levels remained similar, while OPG levels decreased. TNF-alpha inhibition did not influence PG metabolism in cartilage explant culture in the presence of conditioned serum. Although OPG levels were higher and TGF-beta levels were clearly lower in ACS than in SF, intraarticular ACS injection in OA patients did not result in significant changes in these cytokine levels. CONCLUSIONS ACS for treatment of osteoarthritis contains increased levels of anti-inflammatory as well as pro-inflammatory cytokines, in particular TNF-alpha, but conditioned serum does not seem to have a net direct effect on cartilage metabolism, even upon inhibition of TNF-alpha. The fast intraarticular clearance of cytokines in the injected ACS may explain the limited effects found previously in vivo.