To investigate the role of beta-tubulin isotype composition in resistance to paclitaxel, an anti-microtubule agent, human prostate carcinoma (DU-145) cells were intermittently exposed to increasing concentrations of paclitaxel. Cells that were selected and maintained at 10 nM paclitaxel (Pac-10) were fivefold resistant to the drug. Pac-10 cells accumulated radiolabelled paclitaxel to the same extent as DU-145 cells and were negative for MDR-1. Analysis of Pac-10 and DU-145 cells by flow cytometry showed similar cell cycle patterns. Immunofluorescent staining revealed an overall increase of alpha- and beta-tubulin levels in Pac-10 cells compared with DU-145 cells. Examination of beta-tubulin isotype composition revealed a significant increase in betaIII isotype in the resistant cells, both by immunofluorescence and by western blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the isotypes confirmed the increase observed for the betaIII by exhibiting ninefold higher betaIII mRNA levels and also showed fivefold increase of the betaIVa transcript. In addition, analysis of paclitaxel-resistant cells that were selected at increasing levels of the drug (Pac 2, 4, 6, 8 and 10) exhibited a positive correlation between increasing betaIII levels and increasing resistance to paclitaxel. Increased expression of specific beta-tubulin isotypes and subsequent incorporation into microtubules may alter cellular microtubule dynamics, providing a defence against the anti-microtubule effects of paclitaxel and other tubulin-binding drugs.