Single-step selections were used to obtain Chinese hamster ovary cell lines resistant to Colcemid and vinblastine. Verapamil was included in the selections to circumvent the isolation of cells with P-glycoprotein-mediated multidrug resistance and thereby enrich for cells with tubulin alterations. The isolated cell lines were 2-fold resistant to the selecting drug, exhibited cross-resistance to other drugs that inhibit microtubule assembly, and had enhanced sensitivity to the microtubule-stabilizing drug paclitaxel. The concomitant resistance to microtubule-destabilizing drugs and enhanced sensitivity to paclitaxel suggested that these cell lines have changes in microtubule assembly. Consistent with this interpretation, drug-resistant cell lines exhibited altered alpha- or beta-tubulin mobility on two-dimensional gels and higher levels (47-54%) of assembled tubulin compared with wild-type (39%) or paclitaxel-resistant cells (25%). Some drug-resistant cells also had bundled microtubules as judged by immunofluorescence. Genomic sequencing of 11 drug-resistant cell lines predicted five different alterations (D45Y, C211F, D224N, S234N, and K350N) in beta-tubulin and four different alterations (H283Y, E55K, A383V, and R390C) in alpha-tubulin. The amino acid substitutions are dispersed on the primary and tertiary structures of tubulin and, together with the other mutant properties, argue against a mechanism involving changes in drug binding. Rather, we propose that the alterations in alpha- and beta-tubulin increase microtubule stability by promoting longitudinal interdimer and intradimer interactions and/or lateral interactions between protofilaments. This enhanced stability of microtubules increases their resistance to drugs that inhibit assembly.