The development of resistance in trypanosomes to commonly-used trypanocides has an important implication in the chemotherapy and prophylaxis of both human and animal trypanosomosis. This study compared the prophylactic activity of pentamidine isethionate and isometamidium chloride in mice experimentally-infected with Trypanosoma brucei brucei. The 96 mice were divided into four groups with 24 animals each: a) Group I - untreated control group; b) Group II - treated with pentamidine isethionate at 4 mg/kg body weight for 3 alternate days; c) Group III - treated with pentamidine isethionate at 8 mg/kg body weight for 3 alternate days; and d) Group IV - treated once with isometamidium chloride at 1 mg/kg body weight. Thereafter, three mice from each group were infected intraperitoneally with T. brucei brucei at weekly intervals and monitored for parasitaemia. The entire control group was positive for T. brucei brucei by day 6 post infection. Parasitaemia was recorded in Group II at 3 weeks post infection, at 4 weeks in Group III and at 8 weeks in Group IV. Group IV had the highest survival rate followed by Group III. The results showed that isometamidium chloride provides better prophylaxis than pentamidine isethionate.