We investigated the relationships between a putative cofactor of HIV infection, Mycoplasma penetrans, and the evolution of HIV disease. The evolution of titers of anti-M. penetrans antibodies in 58 randomly selected HIV-seropositive adult homosexual men was investigated. The median length of follow-up was 38 months. Thirty-six individuals was investigated. The median length of follow-up was 38 months. Thirty-six individuals (62.1%) remained M. penetrans seronegative (group 0). Fourteen patients (24.1%) had consistently low antibody titers or low antibody titer(s) in at least one sample and negative test(s) in the other(s). This pattern was possibly associated with latent or earlier infection (group 1). Eight patients (13.8%) had moderate to high antibody titers for long periods, indicating an active and persistent M. penetrans infection (group 2); four patients in this group presented a serological reactivation and thus probably developed an acute infection during the study; two had a stable and moderate level of antibody throughout the study; in two patients the antibody titers decreased substantially. Interestingly, CD4 cell counts declined more rapidly in group 2 than in group 0 (medians of -4.5 versus -2.1 cells/mm3/month, p < 0.05 and -0.16 versus 0 cell percentage/month, p < 0.05), whereas there was no significant difference between groups 1 and 0 (medians of -2.0 versus -2.1 cells/mm3/month and -0.15 versus 0 cell percentage/month). In patients with serological reactivation, the viral load was higher in sera with higher M. penetrans antibody titers. These findings suggest an association between active M. penetrans infection and progression of HIV disease.