A set of 26 Trpanosoma brucei stocks from various African countries, previously characterized by multilocus enzyme electrophoresis (MLEE) for 18 polymorphic loci, have been selected to be representative of the three T. brucei classic subspecies. The kinetoplast DNA minicircle variable regions from these stocks have been amplified using the polymerase chain reaction (PCR) technique, and hybridized with the amplified variable regions of three T. brucei reference stocks, previously identified as T. brucei brucei, T. brucei gambiense, and T. brucei rhodesiense, respectively. Both T. b. brucei and T. b. rhodesiense probes hybridized only with their own stocks, but the T. b. gambiense probe specifically hybridized with a group of 12 stocks that represented most of the human stocks from West and Central Africa in our sample. These stocks, which appeared as a clearly separable cluster based on previous MLEE analysis, probably correspond to T. brucei gambiense group I. No other stock hybridized with this amplified fragment. Since the T. b. gambiense probe obtained is specific for many isolates that are pathogenic for humans in Central and West Africa, it appears to be a promising tool for epidemiologic and medical surveys.