Mycoplasma fermentans (incognitus strain) has been linked with acquired immunodeficiency syndrome-associated nephropathy. Ten (23%) of 43 urine sediment specimens from 40 human immunodeficiency virus (HIV)-positive patients at different stages of acquired immunodeficiency syndrome disease tested positive in the polymerase chain reaction using a primer pair found in the insertion sequences specific to M fermentans. Mycoplasma fermentans was isolated from two HIV-positive patients' urine sediment specimens and on a repeated basis from one. All three culture-positive urine sediment specimens tested positive in the polymerase chain reaction. Fifty urine sediment specimens from age-matched HIV-negative healthy controls tested negative for M fermentans by polymerase chain reaction. Mycoplasma fermentans was not isolated from any of the control urine sediment specimens. Our results show a high prevalence of M fermentans in urine sediment specimens from HIV-positive patients but not from urine sediment specimens of HIV-negative healthy controls.