This study investigates the subcellular distribution of microtubules containing acetylated α-tubulin in mammalian cells in culture using the monoclonal antibody 6-11B-1, which specifically recognizes acetylated α-tubulin. The antibody was used in conjunction with B-5-1-2, a monoclonal antibody specific for all α-tubulins, to analyze microtubule structures in 3T3, HeLa, and PtK₂ cells. The results show that acetylated α-tubulin is present in various microtubule structures, including primary cilia, centrioles, mitotic spindles, and midbodies in 3T3 and HeLa cells, but not in PtK₂ cells. These findings confirm that acetylation of α-tubulin occurs in different microtubule structures and in various eukaryotic cells. Acetylated α-tubulin is more resistant to antimitotic drugs and is found in stable microtubules. Taxol treatment increases the acetylation of α-tubulin in 3T3 and HeLa cells, while deacetylation occurs during recovery from exposure to antimitotic drugs or cold. The acetylation of α-tubulin is reversible and may play a role in stabilizing microtubules in specific intracellular spaces. In vitro acetylation of α-tubulin in PtK₂ cells allows the antibody 6-11B-1 to recognize the acetylated epitope, indicating that the absence of the epitope in PtK₂ cells may be due to a lack of acetylation or excessive deacetylation. The acetylation of α-tubulin is a common event in various microtubule structures and is primarily post-assembly. It may be involved in stabilizing microtubules and regulating their assembly. The acetylation and deacetylation of α-tubulin are reversible and may be catalyzed by specific enzymes. The acetylation of α-tubulin is not limited to mammalian cells but is also observed in other eukaryotic cells, including Chlamydomonas. The study highlights the importance of acetylation in microtubule dynamics and its potential role in cellular processes such as cell division and intracellular transport.This study investigates the subcellular distribution of microtubules containing acetylated α-tubulin in mammalian cells in culture using the monoclonal antibody 6-11B-1, which specifically recognizes acetylated α-tubulin. The antibody was used in conjunction with B-5-1-2, a monoclonal antibody specific for all α-tubulins, to analyze microtubule structures in 3T3, HeLa, and PtK₂ cells. The results show that acetylated α-tubulin is present in various microtubule structures, including primary cilia, centrioles, mitotic spindles, and midbodies in 3T3 and HeLa cells, but not in PtK₂ cells. These findings confirm that acetylation of α-tubulin occurs in different microtubule structures and in various eukaryotic cells. Acetylated α-tubulin is more resistant to antimitotic drugs and is found in stable microtubules. Taxol treatment increases the acetylation of α-tubulin in 3T3 and HeLa cells, while deacetylation occurs during recovery from exposure to antimitotic drugs or cold. The acetylation of α-tubulin is reversible and may play a role in stabilizing microtubules in specific intracellular spaces. In vitro acetylation of α-tubulin in PtK₂ cells allows the antibody 6-11B-1 to recognize the acetylated epitope, indicating that the absence of the epitope in PtK₂ cells may be due to a lack of acetylation or excessive deacetylation. The acetylation of α-tubulin is a common event in various microtubule structures and is primarily post-assembly. It may be involved in stabilizing microtubules and regulating their assembly. The acetylation and deacetylation of α-tubulin are reversible and may be catalyzed by specific enzymes. The acetylation of α-tubulin is not limited to mammalian cells but is also observed in other eukaryotic cells, including Chlamydomonas. The study highlights the importance of acetylation in microtubule dynamics and its potential role in cellular processes such as cell division and intracellular transport.