Trypanosoma (Herpetosoma) tamiasi sp. n. is described from blood and organs of the eastern chipmunk, Tamias striatus, and the least chipmunk, Eutamias minimus. In experimentally infected Tamias striatus and E. minimus, Trypanosoma tamiasi reproduced by equal binary fission in the trypo-, sphaero-, epi-, or amastigote form, mainly in lymphoid organs. Trypomastigotes developed from amastigotes through epimastigotes, or from sphaeromastigotes, mainly in the same organs. The mean lengths of body and flagellum of trypomastigotes increased linearly until the organisms reached a maximum length of similar to 37-39 mum. The distance between the nucleus and kinetoplast increased to a mean total length of similar to 33 mum, and thereafter remained constant. Parasitemias of up to 2.5 times 10-7 trypanosomes/ml were produced in Tamias striatus by inoculation with bloodstream, culture, or flea gut forms of Trypanosoma tamiasi; natural parasitemias in Tamias striatus peaked at up to 2.1 times 10-7 trypanosomes/ml. One E. minimus and 2 Spermophilus tridecemlineatus developed parasitemias of more than 1 times 10-6 trypanosomes/ml after inoculation of bloodstream forms of Trypanosoma tamiasi; other experimentally inoculated rodents, including mice, rats, and Spermophilus richardsonii, proved refractory. In chipmunk fleas, Megabothris acerbus, Megabothris quirini, and Tamiophila grandis, bloodstream forms of Trypanosoma tamiasi developed to metacyclic forms in 4-9 days; in rat fleas, Xenopsylla cheopis and Nosopsyllus fasciatus, they survived with little development for up to 2 days, and in kissing bugs, Rhodnius prolixus, up to 10 days. Apparently, the speciation of this trypanosome involved adaptation to certain species of both fleas and rodents.