N-alpha-acetylation, the most common protein modification, involves the transfer of an acetyl group from acetyl-coenzyme A to the N-terminus of a protein or peptide. The major N-alpha-acetyltransferase in Saccharomyces cerevisiae is the ARD1-NAT1 complex. To investigate N-alpha-acetylation in Trypanosoma brucei we have cloned and characterised genes encoding putative homologues of ARD1 and NAT1. Both genes are single copy and ARD1, the putative catalytic component, is expressed in both bloodstream-form and insect-stage cells. In either of these life-cycle stages, disruption of both ARD1 alleles was only possible when another copy was generated via gene duplication or when ARD1 was expressed from elsewhere in the genome. These genetic manipulations demonstrate that, unlike the situation in S. cerevisiae, ARD1 is an essential gene in T. brucei. We propose that protein modification by ARD1 is essential for viability in mammalian and insect-stage T. brucei cells. (C) 2000 Elsevier Science B.V. All rights reserved.