Tubulin is subject to a post-translational acetylation reaction that is thought to be correlated with increased stability of the modified microtubules (MTs). We sought to test directly the stability of acetylated MTs by determining their specific rate of turnover. We used human fibroblasts, which contain a subset of MTs that display terminal and internal domains of acetylation. The turnover of acetylated domains was analysed by microinjecting cells with biotinylated brain tubulin and determining, by triple-label immunofluorescence, the progress of incorporation of biotinylated tubulin into acetylated and non-acetylated domains. Within two minutes after injection, biotinylated domains were contiguous with virtually all observed non-acetylated MT ends but were not contiguous with terminal acetylated domains, demonstrating that the former were growing while the latter were not. Ten minutes after injection, many MTs lacking acetylated domains had incorporated biotinylated subunits uniformly while most MTs containing acetylated domains remained unlabelled, indicating that non-acetylated MTs were turning over while most acetylated domains were not. One hour after injection, virtually all non-acetylated MTs were labelled with biotin whereas approximately half of the acetylated domains contained biotin, demonstrating that acetylated domains turned over much more slowly than the non-acetylated, bulk array. Nonacetylated MT regions flanking acetylated domains also lacked hapten, indicating that acetylation modified discrete regions along stable MTs. Sixteen hours after injection, cells that had not entered mitosis still retained acetylated domains that had not turned over (13% of all acetylated domains), indicating that acetylated domains can be extremely long-lived. Prophase cells displayed no acetylated tubulin staining, indicating that the maximum lifetime of cytoplasmic acetylated domains was one cell cycle. The results show that in this cell line only limited domains of stable MTs usually become acetylated, and that the turnover of these domains and the MTs that bear them is much slower than that of the bulk array.