We prospectively studied 82 men and women with first episodes of genital ulceration. By using newer diagnostic techniques, a definite microbial etiology of 84 infections in 65 of the 82 patients evaluated was found. There were 33 cases of definite primary syphilis, 27 of definite chancroid, and 24 of definite genital herpes simplex. Conclusive evidence of more than one microbial etiology was found for 19 (23%) of the patients. Simultaneous primary syphilis and chancroid was the third most common ulcer infection. This finding underscores the need for both clinical suspicion of multiple infections in patients with genital ulcers and comprehensive testing for all suspicious etiologies.