In three separate surveys involving 413 patients in Mbabane (Swaziland), Nairobi (Kenya), and Carletonville (South Africa), Mycoplasma hominis was isolated from the base of ulcers in 41 (16%) of 251 men with genital ulcer disease. Antibodies to M. hominis were detected by indirect hemagglutination in 89 (51%) of 176 such patients. Of these male patients, 15% and 6% had indirect hemagglutinating antibodies at titers of greater than or equal to 160 and greater than or equal to 640, respectively. The rate of isolation of M. hominis and the results of serologic tests for antibodies to this organism were the same whether or not a cause of genital ulcer disease was identified.