Progress in diagnosis, treatment, and epidemiology of human African trypanosomiasis (sleeping sickness) depends on the existence of specific and sensitive diagnostic tools. Inherent shortcomings of serologic and parasitologic diagnostic methods can be overcome by molecular techniques. Therefore, we have developed a new polymerase chain reaction (PCR) test using primers derived from the recently identified sequence of the Trypanosoma brucei gambiense-specific glycoprotein (TgsGP). The specificity of the TgsGP-PCR was evaluated on DNA extracted from 73 different trypanosome populations belonging to diverse taxonomic groups that were isolated from various host species, and from different geographic origins. The TgsG-PCR was shown to be specific for T. b. gambiense and was suitable for detection of trypanosome DNA in blood samples of patients with confirmed sleeping sickness.