Rheumatoid arthritis (RA), a chronic inflammatory disease, is characterized by hyperplasia of the synovial fibroblasts, due in part to increased cell growth. This study investigated the mechanisms underlying the anti-proliferative action of pectenotoxin-2 (PTX-2), isolated from marine sponges, in synovial fibroblasts obtained from RA patients. PTX-2 concentration-dependently inhibited the growth of synovial fibroblasts, arresting them in the G1 phase of their cell cycle. The G1 arrest was correlated with down-regulation of cyclin D1 and cyclin-dependent kinase (Cdk) 6, with a concomitant up-regulation of the tumor suppressor, p53, and the Cdk inhibitor, p21 (WAF1/CIP1). Following PTX-2 treatment of synovial fibroblasts, an increased binding of p21 with Cdk2 and Cdk6 was paralleled by a significant decrease in retinoblastoma protein (pRB) phosphorylation and in the protein levels of E2F transcription factors. Thus, PTX-2 might help identify new therapeutic agents against RA-mediated hyperplasia of synovial fibroblasts.