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Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and West blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and West blotting.Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression.Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.