Human parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-α), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-α, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-α mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-α promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-α transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-α expression would explain the pathogenesis of B19-associated inflammation.