Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.