To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates (periapical exudates: PE) collected from periapical periodontitis patients were examined. Cell-free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were observed in the supernatants. The production of inducible NO synthase (iNOS) in highly purified PMNs derived from PEs was then immunocytochemically determined using rabbit anti-human iNOS antiserum. In vitro, human peripheral blood PMNs (PB-PMNs) isolated from patients were cultured with a combination of Esherichia coli-lipopolysaccharide (LPS), recombinant human interferon-gamma (rhIFN-gamma) and/or interleukin-1 beta (rhIL-1 beta). The stimulated PB-PMNs showed steady-state levels of nitrite. The stimulation of LPS, rhIFN-gamma and rhIL-1 beta showed more NO induction than that of LPS with either IFN-gamma or IL-1 beta, suggesting the synergistic effects of cytokines. Cryostat sections of surgically removed periapical tissues were also immunohistochemically examined for iNOS, IFN-gamma and IL-1 beta. Two-colour immunohistochemistry revealed the interaction of iNOS-producing PMNs and IFN-gamma- or IL-1 beta-producing mononuclear cells. On the basis of these data, we concluded that with the stimulation of inflammatory cytokines derived from mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothesis suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell-derived cytokines in vivo. NO-producing cells may play a pivotal role in chronic inflammation.