This study analyzed the effect of chondrocyte differentiation on iNOS expression and responses to IL-1 and TGF-beta. During subculturing of chondrocytes, the growth-stimulatory effects of TGF-beta decreased, and cells in later passages even were growth inhibited by TGF-beta. IL-1 beta responses showed an inverse pattern. The antiproliferative effects of IL-1 beta decreased, and, after passage 6, IL-1 beta became a growth stimulator for chondrocytes. This change in growth factor response pattern was associated with a decrease in type II collagen expression. To determine whether these changes in the growth regulatory effects of IL-1 beta and TGF-beta were related to nitric oxide (NO), inducible nitric oxide synthase (iNOS) expression and NO release were analyzed. In primary chondrocytes, TGF-beta did not stimulate iNOS mRNA expression or NO release, and, during co-incubation, it did not detectably alter the IL-1 beta effect. Preincubation with TGF-beta resulted in a time-dependent increase in IL-1-induced NO. With increasing passage number, the IL-1 beta effects decreased, and, after passage 6, IL-1 beta no longer detectably stimulated iNOS expression or NO release. However, TGF-beta increased NO production synergistically with IL-1 beta during the same culture period when it lost its growth-stimulatory effects. The antiproliferative effects of TGF-beta in late passage chondrocytes were reversed by the NO synthase inhibitor NG-monomethylarginine. These results suggest a novel pattern of iNOS regulation by IL-1 and TGF-beta and show that the factors that modulate iNOS expression and proliferation are dependent on the differentiation status of the cells.