Phosphonylmethoxyalkylpurines and -pyrimidines exhibit potent activity against a broad spectrum of DNA viruses. We evaluated some of these nucleotide analogues for antitrypanosomal activity in vitro and in mice. The most active compounds were (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine (HPMPDAP), which inhibited growth of Trypanosoma brucei brucei by 50% (EC50 value) when incubated in vitro for 24 hr with 0.23-5.69 micrograms drug/ml. Both compounds completely eliminated multidrug-resistant T. b. brucei in culture at 1 microgram/ml after 4-5 days exposure. Mice infected with drug-susceptible T. b. brucei were cured with 2 doses of 10 mg/kg HPMPDAP. Two or 5 doses of 50 mg/kg 9-(2-phosphonylmethoxyethyl) adenine (PMEA) or 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), respectively, were necessary to eliminate T. b. brucei infections in mice. Mice infected with multidrug-resistant T. b. brucei were not cured with the above dosages. The most active compound against Trypanosoma congolense was PMEDAP with an EC50 value of 3.21-11.63 micrograms/ml. Thus, some of the phosphonylmethoxyalkyl purines showed potential as antitrypanosomal compounds at dosages that are below those toxic for mice.