Flagellate infections were found in 1,063 of 18,895 sand flies collected in the states of Amazonas, Pará, Rondonia and Acre, Brazil. Infection rates were 13.4% (species group Shannoni); 7.5% (subgenus Nyssomyia); 6.7% (subgenus Lutzomyia series Cruciata); 0.5% (genus Psychodopygus) and 3.1% for other sand flies (various subgenera). Leishmania braziliensis guyanensis and L. mexicana amazonensis were isolated, respectively, from the known vectors, Lutzomyia umbratilis and L. flaviscutellata. Single stocks of L. braziliensis-like and L. mexicana-like organisms were isolated, respectively, from L. whitmani and L. yuilli. Thirty-eight flagellate stocks, isolated by direct culture from sand flies were characterized in detail by morphology in culture, behavior in hamsters and mice and by enzyme profiles. Sixteen stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum schaudinni; 8 stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum sp.; 7 stocks from Psychodopygus ayrozai and P. paraensis were identified as Leishmania sp. previously isolated from the armadillo, Dasypus novemcinctus; 2 stocks of Trypanosoma rangeli were isolated from recently fed Lutzomyia sp. (Shannoni group) sand flies; the remaining 5 stocks from L. umbratilis and L. yuilli could not be identified. Observations suggested that Shannoni group sand flies were the natural vectors of Endotrypanum. Leishmania sp. infections in the man-biting flies P. ayrozai and P. paraensis were restricted to the midgut and associated with recent bloodmeals. Unidentified flagellates in L. umbratilis and L. yuilli were distributed throughout the digestive tract with no trace of bloodmeals.