We examined files of various families for the presence of trypanosomatids. Of 592 insects, 113 (19%) were positive. From these insects, we obtained 42 cultures and selected 14 for further analysis. Seven of the cultures had the characteristics of Herpetomonas species, they displayed typical opisthomastigotes, lacked arginase, possessed a Pvu II restriction site at 360 bp from the 5' end of the small subunit ribosomal gene, and did not possess a Hin dIII site at 1,500 bp from the ribosomal alpha-large subunit 5' end. Hybridization with synthetic oligonucleotides complementary to the sequences flanking the Pvu II site in Herpetomonas samuelpessoai and Herpetomonas muscarum, permitted the distribution of the old species and the 7 isolates into 2 subgroups of Herpetomonas spp. Of the remaining 7 cultures, 4 were probably Leptomonas spp., whereas the other 3, together with Herpetomonas roitmani, seem to constitute a novel group with morphological and molecular characteristics quite distinct from those of Herpetomonas spp. or any other genera of Trypanosomatidae. We also studied some trypanosomatid species of questionable taxonomic status, Leptomonas samueli and Phytomonas davidi yielded results identical to those of Herpetomonas spp., thus confirming their already suspected affiliation to this genus. On the other hand, Herpetomonas anglusteri, Herpetomonas dedonderi, and Herpetomonas mcgheei displayed morphological and molecular characteristics incompatible with their placement in the genus Herpetomonas.