Nitric oxide (NO) appears to play a key role in the pathogenesis of Gram-negative bacterial sepsis. In inflammatory conditions, NO production is dependent upon an enzyme known as the inducible nitric oxide synthase (iNOS). Little is known regarding the potential involvement of iNOS/NO in Grampositive sepsis. We tested the ability of pneumococci, pneumococcal cell wall preparations (PCWPs), and purified pneumococcal polysaccharide capsule to trigger the production of iNOS and NO in RAW 264.7 murine macrophages. Immunoblotting was utilized to measure iNOS protein levels in cell lysates, while NO production was estimated by determination of nitrite concentrations in cell culture supernatants by the Griess reaction. We examined two strains of Streptococcus pneumoniae --strain 6303, obtained from the ATCC, and strain 124, a clinical isolate from a child with sepsis; results with both strains were similar. We found that live pneumococci, oxacillin-killed pneumococci, and PCWPs stimulated the production of both iNOS and NO in RAW 264.7 cells in the presence (but not the absence) of low concentrations of recombinant murine interferon-gamma (rIFN γ). In contrast, purified pneumococcal capsule (Pnu-Imune 23, Lederle Laboratories) failed to induce either iNOS or NO production in the presence or absence of rIFN γ in these cells. Polymyxin B, a potent inhibitor of endotoxin, blocked iNOS and NO production in RAW 264.7 cells exposed to rIFN γ + LPS but not rIFNγ + pneumococci or PCWPs. We conclude that pneumococci stimulate the production of both iNOS and NO by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.