Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non-inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.