Turnover of tyrosinated and detyrosinated microtubules ([Tyr]MTs and [Glu]MTs, respectively) was analyzed by the combined use of hapten-mediated immunocytochemistry and peptide-specific antibodies. Cells were microinjected with hapten-labeled tubulin and then processed for triple-label immunofluorescence to determine the pattern of incorporation of the injected subunits into [Tyr]- and [Glu]-MTs. Within 2 min of microinjection, hapten-labeled domains were present at the ends of virtually all [Tyr]MTs but were absent from most [Glu]MTs, demonstrating that [Tyr]MTs grew, whereas most [Glu]MTs did not. After 1 hr of incubation, all [Tyr]MTs analyzed were copolymers of endogenous and hapten-labeled subunits, indicating complete and rapid turnover of these MTs. However, the majority of [Glu]MTs were not hapten-labeled, indicating that they had not turned over. Even 16 hr after injection, cells that had not divided retained a small proportion of [Glu]MTs lacking hapten, implying that some had persisted for most of a cell generation. At mitosis, all MTs were hapten-labeled, indicating that the stable interphase [Glu]MTs had depolymerized. The results establish that the MT network is heterogeneous in its turnover rate, being composed of at least two populations: [Tyr]MTs that turn over rapidly and [Glu]MTs that turn over slowly.