Acetylation is the most frequently occurring chemical modification of the α-NH2 group of eukaryotic proteins and is catalyzed by a Nα-acetyltransferase. Two-dimensional gel electrophoresis was used to compare the soluble proteins synthesized in wild type and a mutant (aaal) yeast cells lacking Nα-acetyltransferase. Among 855 soluble proteins identified in wild type and mutant, ~20% of the proteins in the mutant either disappeared or were shifted to higher pI without a change of molecular mass, and 27 proteins were observed only in the mutant. In addition, the synthesis of another 12% of the proteins in the mutant was either diminished or enhanced, suggesting that the acetylation of certain regulatory proteins may affect their expression. This is the first demonstration of the broad-based functional role of Nα-acetylation in eukaryotic protein synthesis. Acetyltransferase; Acetylation; (Saccharomyces cerevisiae). © 1989.