The purpose of this investigation was to determine whether mouse lung fibroblast subsets have the ability to produce nitric oxide (NO), and if so, to characterize the induction and effects of its synthesis. Previously, we isolated Thy1+ and Thy1- subpopulations of mouse lung fibroblasts, which differ in terms of cytokine production, morphology, response to cytokines and radiation, and ability to present antigen to T lymphocytes. When treated with the proinflammatory cytokines IFN-gamma, TNF-alpha, and IL-1 alpha, these fibroblast lines produce micromolar quantities of NO2- and NO3-, two stable end products of the NO pathway. A combination of all three cytokines provided the greatest induction, and there was no measurable production of NO in unstimulated cells. Thy1+ fibroblasts have fewer requirements for induction of NO production than the Thy1- line, in that NO production could be induced by only two of the above cytokines, where the Thy1- fibroblasts required all three. Inducible NO synthase (iNOS) mRNA was shown to be present by the reverse transcriptase-polymerase chain reaction as early as 2 hr after cytokine treatment in both cell lines. Addition of the NO synthase inhibitors NG-monomethyl-L-arginine and aminoguanidine inhibited production of NO2- and NO3-, but not iNOS mRNA. This inhibition was partially reversed by the addition of an excess of L-arginine. Interestingly, inhibition of NO synthesis was shown to decrease IL-6 production by more than 50% in cytokine-treated Thy1+ fibroblasts. These results indicate for the first time that Thy1+ and Thy1- mouse lung fibroblast subsets have the capability to produce NO to differing extents in response to cytokines and may therefore play an important role in the inflammatory response in the lung as well as in the progression of lung disease.