Trypanosome lytic factor (TLF) provides innate protection for humans against infection by the animal pathogen Trypanosoma brucei brucei but not against the agent of human African sleeping sickness, Trypanosoma brucei rhodesiense. TLF exists in two forms, TLF-1 and TLF-2. Prior studies suggested that TLF-1 causes lysosomal disruption and subsequent cell death in T. b. brucei. Here we confirm the lysosomal targeting of TLF-1 by immunolocalization with the trypanosome lysosomal membrane protein p67, and by co-fractionation of radiolabelled TLF-1 with lysosomal enzymes. In addition, pulse-chase studies indicate that TLF-1 is not degraded within the lysosome as compared to the host protein transferrin. In TLF-1 treated cells, transferrin is degraded normally, indicating that lysosomal proteases remain active during the early phase of TLF-1 treatment but fail to degrade TLF-1. Following endocytosis a TLF lipoprotein appears to undergo disulfide bond reduction prior to entering the lysosome. Results presented here indicate that TLF-1 lipoproteins are targeted to the lysosome but are resistant to trypanosome lysosomal proteases.