In two studies Mycoplasma hominis was isolated from the upper urinary tracts of seven of 80 patients with symptoms of acute pyelonephritis and three of 18 patients with signs of acute exacerbation of chronic pyelonephritis. M. hominis was not recovered from the upper urinary tract of 22 patients with chronic pyelonephritis without acute exacerbation or from 60 patients with noninfectious urinary tract disease. In an additional study, specimens were cultured from patients with antibodies to M. hominis in urine. All of these patients had acute pyelonephritis; M. hominis was isolated from the upper urinary tract of seven patients. Thus, M. hominis was cultured from the upper urinary tract of a total of 17 patients. No other microorganisms were recovered from 12 of these patients. The titer of antibody to M. hominis significantly increased or decreased in the serum of 13 patients, and antibodies were demonstrated in the urine of nine. These results suggest that M. hominis may be a cause of acute pyelonephritis in humans; the organism is estimated to account for approximately 5% of such cases. The clinical signs of infection in the patients studied were subtle, and the symptoms did not involve the lower urinary tract. The diagnosis of acute pyelonephritis due to M. hominis can be established by culture of the organism from the upper urinary tract and its likely if antibodies are present in urine; in contrast, culture of M. hominis from catheter-collected urine or detection of a serum antibody response is not sufficient evidence for this diagnosis.