The polymerase chain reaction (PCR) method was used to characterize trypanosome infections in tsetse flies from 3 sleeping sickness foci in Cameroon. The predominant tsetse species found was Glossina palpalis palpalis. An average infection rate of 12.1% was revealed by microscopical examination of 888 non-teneral tsets flies. PCR amplification analyses for trypanosome identification were carried out on 467 flies, with primer sets specific for Trypanosoma (Trypanozoon) brucei s.1., T. (Duttonella) vivax, T. (Nannomonas) simiae and forest type T. (Nannomonas) congolense. Of 467 flies 93 were positive by microscopical analysis while PCR succeeded in identifying 89 positive flies. Of the PCR-positive flies 34 (38.2%) were negative by microscopical examination. PCR amplification, when compared to the parasitological technique, gave a higher estimate of infection rate of trypanosomes in natural tsetse populations. The PCR technique did, however, fail to identify 40.9% (38/93) of the parasitologically positive flies. The reasons for this failure are discussed. The overall prevalence of mixed infections, assessed by PCR, was 37.1%; the majority (72.7%) involved T. brucei and forest type T. congolense.