It was found previously that human immunodeficiency virus type 1 (HIV-1)-irrelevant CD8+ cytotoxic T lymphocytes (CTLs) from uninfected donors suppressed HIV-1 replication in a cell-contact-dependent manner. However, one of these CTL lines (CTL-3) also significantly suppressed HIV-1 replication through its supernatant. Here, the suppressive fraction from CTL-3 supernatant was purified and analysed by mass spectrometry. A protein band specific for the suppressive fraction was identified as arginine deiminase from Mycoplasma arginini, which catalyses the hydrolysis of arginine to citrulline. Addition of L-arginine or the use of antibiotics against mycoplasma restored supernatant-mediated but not cell-contact-dependent suppression of HIV-1 replication by CTL-3, clearly indicating that arginine deiminase of M. arginini in the supernatants suppressed HIV-1 replication, which is independent of CD8+ T-cell-mediated HIV-1 suppression via cell contact. Arginine deiminase is known to be a chemotherapeutic agent against arginine-requiring tumours and these results suggest that it also has potential application in antiviral therapy.