A total of 498 two-toed sloths, Choloepus hoffmanni, collected in central Panama was examined for Leishmania braziliensis over a 10-year period. Isolations of the parasite from 96 (19.3%) of the animals were confirmed by culture and inoculation of golden hamsters. Improved culture techniques developed toward the end of the study assisted in determining a greater prevalence of the disease. Infections were completely cryptic in all animals, and the parasite was isolated from skin, blood, liver, spleen, bone marrow and lung tissues. Sloths maintained under seminatural conditions remained infected up to 23 months, the longest period of survival. This edentate, considered the principal reservoir host of L. braziliensis in Panama, showed infection rates from 0–59.4% in various communities, which appeared to correlate with the parasite prevalence in the indigenous human populations.