The 5 cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m 7 GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m 7 GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3 untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m 7 GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.Trypanosomatids are ancient eukaryotes that cycle between invertebrate vectors and mammalian hosts, causing a wide range of diseases. Leishmania parasites exist as extracellular flagellated promastigotes in the alimentary canal of female flies, and upon transfer to the mammalian host, they enter macrophages and cells of the immune system, transforming into amastigotes. Leishmania parasites are exposed to a broad range of environmental conditions, and stage differentiation is triggered by changes in temperature and pH (22,57).Trypanosomatids are characterized by a variety of unique molecular features, including polycistronic transcription of protein-coding genes (48, 60) and trans splicing (37), whereby a small leader RNA of 39 nucleotides, denoted spliced leader RNA (SL RNA), is spliced onto the 5Ј ends of all mRNAs, providing the cap structure. The trypanosomatid cap is a highly modified structure that, in addition to m 7