Post-translational modifications (PTMs) of histones regulate almost all facets of DNA metabolism in eukaryotes, such as replication, repair, transcription and chromatin condensation. While histone PTMs have been exhaustively examined in yeast and higher eukaryotes, less is known of their functional consequences in trypanosomatids. Trypanosome histones are highly divergent from those of other eukaryotes, and specific PTMs have been identified in histones of Trypanosoma species. The characterization of three MYST-family histone acetyltransferases (HATs) in Trypanosoma brucei had earlier identified the HATs responsible for acetylation of two lysine residues, K4 and K10, in the N-terminal tail of histone H4. This report presents the results of what we believe to be the first study of a HAT in a Leishmania species. The HAT4 gene of Leishmania donovani, the causative pathogen of visceral leishmaniasis, was cloned and expressed in fusion with GFP in Leishmania promastigotes. We found that HAT4-GFP behaves differently from typical eukaryotic MYST-family HATs, which are usually constitutively nuclear, in that it is cytosolic throughout the cell cycle, although the protein is also present in the nucleus in post-mitotic cells. Substrate-specificity analyses revealed that LdHAT4 acetylates the N terminus of histone H4, but not those of H2A, H2B or H3. Nor does it acetylate the C terminus of H2A. The primary target of HAT4-mediated acetylation is the K14 residue of H4, although K2 may be a minor site as well. H4K14 acetylation in Leishmania may occur either in the cytoplasm prior to histone deposition, or soon after mitosis in the nucleus.