Interferon production was measured in the murine mixed lymphocyte tumor cell interaction (MLTI) using spleen cells of three inbred mouse strains and a number of in vitro grown lymphoma cells. No difference in interferon production was observed when syngeneic or allogeneic combinations were compared. Interferon was not detectable during the first 6 h of culture and reached its maximum after 12--24 h. In contrast, natural killer cell activity of spleen cells against one of these lymphoma lines (YAC-1) was already high after 4h. A cell line that was not susceptible to natural killing (MDAY-D2) induced the same amount of interferon as YAC-1. These findings suggest that natural killer cell activity and interferon induction in the MLTI are not correlated. Several lymphoma cell lines induced interferon in the MLTI whereas some others did not. All lines that were inductive were found to contain mycoplasmas. Furthermore, even the cell-free supernatants of inducing lines contained mycoplasmas and induced interferon. Purified mycoplasmas and membranes thereof were able to induce interferon production in mouse spleen cells. Our data strongly suggest that interferon production in the MLTI is caused by mycoplasmas. This artifact is the relevance for three reasons. First, cocultures between lymphoma cells and lymphocytes are widely studied in immunology. Secondly, contamination with mycoplasmas is extremely common and often goes unnoticed in immunological laboratories. Thirdly, interferon is known to affect profoundly a number of in vitro functions of immunocompetent cells.