Following the discovery of acetylated alpha-tubulin in the flagella of Chlamydomonas, many studies have documented the presence of acetylated alpha-tubulin in a variety of evolutionarily divergent organisms. While this posttranslational modification may define an isoform with a unique function, the primary effect of alpha-tubulin acetylation remains unknown. To study the function of alpha-tubulin acetylation, we have transformed Chlamydomonas, an organism in which almost all of the flagellar tubulin and a subset of the cytoplasmic microtubules are acetylated, with an alpha 1-tubulin gene whose product cannot be acetylated. Specifically, the codon for lysine 40, the lysine that is acetylated, has been replaced with the codon of nonacetylatable amino acids. To distinguish mutagenized alpha-tubulin from that produced by the two endogenous alpha-tubulin genes, mutant alpha-tubulin was tagged with an epitope from influenza virus hemagglutinin. Utilizing the constitutive Chlamydomonas rubisco small subunit S2 promoter, we have obtained in selected clones high levels of nonacetylatable alpha-tubulin expression approximating 50-70% of the total flagellar alpha-tubulin. Immunofluorescence and immunoblot analysis of transformed cells indicated that nonacetylatable alpha-tubulin could assemble, along with endogenous alpha-tubulin, into both cytoplasmic and flagellar microtubules. However, no gross phenotypic effects were observed, suggesting that the effect of alpha-tubulin acetylation is subtle.