Zymosan-induced peritonitis represents a well-described model of acute inflammation. The binding of zymosan with its specific Toll-like receptors (TLR2 and TLR6) on leukocytes initiates activation and phosphorylation of nuclear factor (NF)-κB, which leads to accumulation of NF-κB p65 subunits in the nucleus and subsequently up-regulation of the proinflammatory cytokine genes expression. Intraperitoneal co-administration of zymosan and morphine significantly inhibits peritonitis in several strains of mice by decreasing the influx of exudatory cells; however, mechanisms of this action still remain unclear. We aimed to verify the effects of morphine on NF-κB and TLRs expression at messenger RNA and protein levels during the early stages of zymosan-induced peritonitis. Peritonitis was induced by a single injection of zymosan A or zymosan supplemented with morphine in Swiss mice. At selected time points, after stimulation, peritoneal leukocytes were harvested. The TLRs and NF-κB expression was assessed by real-time PCR and flow cytometry. In comparison with the mice injected with zymosan only, morphine co-injection significantly decreased the expression of phospho-NF-κB and TLR2 in all investigated immunocompetent cells as well as up-regulated the levels of nitric oxide (NO) in peritoneal fluid. Moreover, supplementation of zymosan with morphine altered the TLR, NF-κB and some proinflammatory cytokines (keratinocyte-derived chemokine, tumor necrosis factor-α) gene expression during ongoing inflammation. We may postulate that after morphine stimulation peritoneal leukocytes recognize less effectively zymosan antigens because of impaired TLRs expression. The lower TLR expression attenuates TLR-mediated signal transduction, which prevents NF-κB activation. Additionally, during zymosan-induced peritonitis, morphine may modulate the NF-κB expression, at least partially, by an up-regulated release of NO, as suggested by others.