Trypanosoma brucei has telomeres composed of 15-kb tracts of TTAGGG repeats that end in 3´ overhangs and form t-loops. This structure is also present in mammalian cells and is thought to reflect the presence of telomere-binding proteins. The human TTAGGG repeat-binding factor TRF1 binds to telomeres and regulates their length. We attempted to interfere with the normal function of trypanosome telomeres by expressing human TRF1 in T. brucei. TRF1 localized to telomeres in cultured procyclic (midgut-stage) trypanosomes with great fidelity, but not in bloodstream-stage trypanosomes. Procyclic trypanosomes expressing high levels of TRF1 for extended periods of time exhibited shortening and increased size heterogeneity of their telomeres and the cell cycle was arrested in G1&endash;S. These effects were not detected in cells expressing a TRF1 mutant incapable of binding to TTAGGG repeats. We argue that TRF1 displaces putative endogenous trypanosome telomere-binding proteins, not yet identified, and affects telomeres in ways that reflect its role as a negative regulator of telomere length in human cells. (C) 2001 Elsevier Science B.V. All rights reserved.