Voided urine samples from persons with and without human immunodeficiency virus (HIV) infection were examined for mycoplasmas. Mycoplasma hominis organisms were identified in cultures of urine from 32 (18%) of 180 HIV-positive individuals and from 8 (21%) of 38 HIV-negative individuals. In contrast, glucose-utilizing mycoplasmas were identified in the urine of 30 (17%) of the HIV-positive individuals and in none of those who were HIV-negative. Assays of growth inhibition around disks containing specific antisera identified 14 of the 30 glucose-utilizing mycoplasmas as Mycoplasma fermentans. Four isolates were presumptively identified as Mycoplasma pirum. Growth on solid media was insufficient to permit the identification of the species of the other 12 isolates by growth inhibition.