The relative importance of Haemophilus ducreyi and Treponema pallidum in genital ulcer disease in Africa has decreased recently, whereas that of herpes simplex virus (HSV) type 2 has increased. We analysed 301 lesional specimens from Tanzanian patients with genital ulcer disease for the presence of H. ducreyi, T. pallidum and HSV-1/HSV-2 by performing a separate PCR for each pathogen. Infectious agents were detected in 211 (70%) of the cases. A single pathogen was found in 191 samples and two or more pathogens in the remaining 20. HSV-2 represented 83% of all identified pathogens, HSV-1 8%, T. pallidum 4% and H. ducreyi 5%. HSV-1 was identified as a single pathogen in four samples, in combination with others in an additional 14 samples. Thus, HSV-1 can also be the cause of genital ulcer disease in Africa. Regular surveillance of genital ulcer disease aetiology is important in programs for management of genital ulcer disease and HIV in Africa.