To investigate the mechanisms of action underlying the anti-inflammatory effects of the nonsteroidal anti-inflammatory drug aceclofenac in humans, we studied the metabolism of aceclofenac in detail in primary cultured synovial cells of 10 patients with rheumatoid arthritis. Aceclofenac and 4'-hydroxyaceclofenac are the major compounds in human blood after the administration of aceclofenac, but they had no inhibitory effects on cyclooxygenase (COX) activity or COX expression in the rheumatoid synovial cells. In contrast, aceclofenac and 4'-hydroxyaceclofenac reduced prostaglandin E2 (PGE2) production by the rheumatoid synovial cells. We also observed that aceclofenac and 4'-hydroxyaceclofenac were hydrolyzed into the COX inhibitors diclofenac and 4'-hydroxydiclofenac, respectively, by the rheumatoid synovial cells. However, the hydrolytic activity differed markedly among the cell preparations. Because the suppressive potency of aceclofenac and 4'-hydroxyaceclofenac against the PGE2 production was proportionally correlated with the hydrolytic activity in rheumatoid synovial cell preparations, we suggest that the suppressive effects of aceclofenac and 4'-hydroxy aceclofenac on PGE2 production are facilitated by the hydrolytic activity in rheumatoid synovial cells.