Gene expression in the Leishmania is controlled post-transcriptionally, and is likely to be impacted by both 5′ and 3′ untranslated regions (UTRs). We have investigated the effects of trinucleotides in the AUG-proximal region (APR) (i.e. positions −3 to −1 upstream of an AUG) on two reporter genes in the context of an endogenous intergenic region of Leishmania tropica. The effects of APRs on protein expression were determined in stable transfectants in vivo. Three APRs, namely, C−3C−2C−1, ACC and GCC, yielded robust translation, whereas GTA produced low amounts of proteins. A purine at −3 of an APR was not crucial for efficient translation. Steady-state level of reporter mRNA did not correlate directly with the amount of protein detected. Polysome analysis revealed that APRs modulate translation, at least in part, by influencing mRNA association with ribosomes. An analysis of genomic UTRs in L. major showed that (i) the consensus APR is N−3N−2C−1 (where N = any nucleotide), and (ii) the most frequently used APRs include ACA, ACC, ATC, GCC, GCG, GTC and CAC, some of which were translation enhancers in our experimental studies.