In addition to previously reported tetracycline analogs, other antibiotics known for antimycoplasmal activities inhibited the cytopathic effect in CEM cl13 cells infected with human immunodeficiency virus type 1 (HIV-1) or HIV-2 but were unable to block virus replication. A contaminating mycoplasma was isolated from our CEM cl13 cells and identified as a strain of Mycoplasma fermentans. Following infection of lymphoblastoid (CEM) or promonocytic (U937 and THP1) cell lines with HIV-1, cytopathic effect was observed only in association with mycoplasmal contamination. Moreover, HIV-1 infection of U937 cells after experimental inoculation with a human isolate of M. fermentans led to pronounced cell killing. We have verified that this effect is not merely an artifact caused by arginine and/or glucose depletion in the cell culture medium. These results confirm that mollicutes, in particular M. fermentans, are able to act synergistically with HIV-1 to kill infected cells in some in vitro systems.