The aim of this study was determine the prevalence of Mycoplasma hominis, M. genitalium, M. fermentans, M. pirum, M. penetrans and Ureaplasma urealyticum in HIV-infected patients. Culture and PCR were used to detect six species of Mycoplasma in first-void urine of HIV-1 infected men. A total of 497 HIV/AIDS patients (age range 5-75 years, mean 37 years) were screened in the study. All presented positive for at least one kind of mycoplasma, especially U. urealyticum and M. hominis. Six mycoplasmas were significant in the homosexual contact and heterosexual contact groups. The distribution of M. hominis, M. penetrans, and M. pirum were significantly different in this four-transmission category. CD4+ cell count levels were lower in the AIDS-associated Mycoplasma-positive group than in the Mycoplasma-negative group (P<0.01). This study indicates that U. urealyticum, M. hominis and M. fermentans are prevalent in HIV-1-infected male patients. This may be an indication of whether mycoplasmas are co-factors in the progression of HIV disease.