Several different Mycoplasma species have been shown to act as mitogens for either T or B cells and as stimulators of macrophage tumoricidal activity. In this report, we show that at least five different species of Mycoplasma are capable of inducing class II MHC expression on macrophages. We have observed significant induction of class II MHC surface expression on the myelomonocytic cell line, WEHI-3, as early as 24 h after deliberate infection of cultures, reaching maximal levels by 4 days. This induction was also apparent at the mRNA level as assessed by Northern blot analysis by using A alpha, E alpha, and A beta probes. However, unlike many other previously described MHC-inducing agents, mycoplasmas failed to induce class I MHC expression at either the cell surface or mRNA levels. Kinetic analysis revealed that induction of class II mRNA by mycoplasmas was slower than induction by IFN-gamma requiring 24 h rather than 8 h for significant increases to be noted. Induction by mycoplasmas does not require the presence of live organisms and remains active after heat treatment of 90 degrees C for 30 min. We have also demonstrated that mycoplasma infection of primary bone marrow macrophage cultures leads to the induction of both class I and class II genes and, as in the case of WEHI-3, this induction does not require the presence of live organisms. These data indicate that several Mycoplasma species have the capacity to induce class II MHC expression in WEHI-3 and both class I and class II MHC expression in bone marrow macrophage cultures in the absence of any T cell products.