ABSTRACT Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP ( Lm PABP) homologues: the previously described Lm PABP1; Lm PABP2, orthologous to the PABP described from Trypanosoma species; and Lm PABP3, unique to Leishmania . Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, Lm PABP1 binding was poly(A) sensitive but heparin insensitive; Lm PABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and Lm PABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only Lm PABP1 is present as multiple isoforms. Upon transcription inhibition, Lm PABP2 and -3 migrated to the nucleus, while Lm PABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between Lm PABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) ( Lm EIF4G3) in vitro , Lm PABP1 was the only one to copurify with native Lm EIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei , where both orthologues to Lm PABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for Lm PABP1 in translation through its interaction with the eIF4G homologue.