Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNF alpha) production in THP-1, U937 and J22HL60 cell lines, and in the enhancement of HIV-1 replication in a dormantly-infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh-Dyer method. TNF alpha production was observed in the peritoneal macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNF alpha production and enhancement of HIV-1 replication were strongly inhibited by Concanavalin A-Sepharose. The inhibitory effect of Concanavalin A-Sepharose was partially prevented by sugars in the order methyl-alpha-D-mannopyranoside and methyl-alpha-D-glucopyranoside but not methyl-alpha-D-galactopyranoside. Anti-human TNF alpha antibody, however, did not reduce the activity of the methanol layer to enhance HIV-1 replication, suggesting that the methanol layer could enhance HIV-1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV-1 infection.