This study investigates the subcellular distribution of microtubules containing acetylated α-tubulin in mammalian cells in culture using a monoclonal antibody, 6-11B-1, specific for acetylated α-tubulin. The antibody was used to stain 3T3, HeLa, and PtK2 cells, and the results were compared with those obtained using a second antibody, B-5-1-2, which recognizes all α-tubulin isoforms. The study found that acetylated α-tubulin is present in microtubules of 3T3 and HeLa cells but not in PtK2 cells. These microtubules are associated with primary cilia, centrioles, mitotic spindles, midbodies, and specific segments of cytoplasmic microtubules. The acetylation of α-tubulin was also observed in microtubules stabilized by taxol, suggesting that acetylation occurs after microtubule assembly. Additionally, drug-resistant microtubules, which are more stable under conditions promoting microtubule disassembly, contained more acetylated α-tubulin than other cytoplasmic microtubules. The acetylation of α-tubulin was found to be reversible, and the antibody 6-11B-1 did not bind to acetylated α-tubulin in PtK2 cells, indicating that the epitope recognized by the antibody is absent or masked in these cells. The study concludes that acetylation of α-tubulin is a common event in various microtubule structures and may play a role in regulating the stability and organization of microtubules.This study investigates the subcellular distribution of microtubules containing acetylated α-tubulin in mammalian cells in culture using a monoclonal antibody, 6-11B-1, specific for acetylated α-tubulin. The antibody was used to stain 3T3, HeLa, and PtK2 cells, and the results were compared with those obtained using a second antibody, B-5-1-2, which recognizes all α-tubulin isoforms. The study found that acetylated α-tubulin is present in microtubules of 3T3 and HeLa cells but not in PtK2 cells. These microtubules are associated with primary cilia, centrioles, mitotic spindles, midbodies, and specific segments of cytoplasmic microtubules. The acetylation of α-tubulin was also observed in microtubules stabilized by taxol, suggesting that acetylation occurs after microtubule assembly. Additionally, drug-resistant microtubules, which are more stable under conditions promoting microtubule disassembly, contained more acetylated α-tubulin than other cytoplasmic microtubules. The acetylation of α-tubulin was found to be reversible, and the antibody 6-11B-1 did not bind to acetylated α-tubulin in PtK2 cells, indicating that the epitope recognized by the antibody is absent or masked in these cells. The study concludes that acetylation of α-tubulin is a common event in various microtubule structures and may play a role in regulating the stability and organization of microtubules.