The anti-cancer drug taxol binds to beta-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC50 greater than 11.7 microM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 microM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding beta-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of beta-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of beta-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [3H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [3H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various beta-tubulin sequences showed that beta-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but beta-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and beta-tubulin.