Peroxin 13 (PEX13) is one of the components of a peroxisomal membrane complex involved in import of proteins into the matrix of the organelles and has previously been characterized in a variety of organisms. Trypanosomatids (Trypanosoma, Leishmania), protozoan parasites having peroxisome-like organelles designated glycosomes, possess an unusual PEX13 which shares very low sequence identity with others and lacks some typical PEX13 characteristics. It was identified in the databases through its multiple YGx motifs present in a glycine-rich N-terminal region of low sequence complexity. Like other PEX13s, it contains predicted transmembrane segments and a SH3 domain in its C-terminal half. The localization of T. brucei PEX13 in the glycosomal membrane was confirmed by expression of a fusion construct with Green Fluorescent Protein, and western blot analysis of purified organelles and membranes. The C-terminal half of the protein was shown to interact with the third of three pentapeptide repeats of the previously characterized PEX5, the receptor of glycosomal proteins with a type 1 peroxisome-targeting signal, and with PEX14, another component of the same peroxisomal protein import complex in the membrane. PEX13 is essential for the parasite; depletion by RNA interference results in mislocalization of glycosomal proteins and death of the parasites.