The objective of this study was to investigate the stability and the degradation pathway of epothilone-D (Epo-D), an experimental anticancer agent. In pH range 4-9, Epo-D displayed pH-independent stability and the highest stability was observed at pH 1.5-2 where its thiazole group is protonated. Increasing the pH >9 or <1.5 resulted in an increase in the degradation rate. Epo-D contains an ester group that can be hydrolyzed. The formation of the hydrolytic product was confirmed by the nuclear magnetic resonance (NMR), fast atom bombardment mass spectroscopy and liquid chromatography/mass spectroscopy/mass spectroscopy techniques. The largely sigmoidal pH-rate profile is not consistent with the normal pH dependency of ester hydrolysis involving an addition/elimination mechanism. Hence, a hydrolysis mechanism through a carbonium ion was suggested. At pH 4 and 7.4, no buffer catalysis was observed (0.01, 0.02, and 0.05 M buffers) and no significant deuterium kinetic solvent isotope effect was noted. The degradation was very sensitive to changes in the dielectric constant of the solvents as significant enhancement in the stability was observed in buffer-acetonitrile and 0.1 M (SBE)7m-beta-cyclodextrin solutions compared with just buffer, suggesting that the rate-determining step in the degradation pathway involved formation of a polar transition state. Mass spectral analysis of the reaction run in 18O water was consistent with incorporation of the 18O in the alcohol hydroxyl rather than the carboxylate group. These observations strongly support the carbonium ion mechanism for the hydrolysis of Epo-D in the pH range 4-9. A pKa value of 2.86 for Epo-D was estimated from the fit of the pH-rate profile. This number was confirmed independently by the changes in ultraviolet absorbance of Epo-D as a function of pH (pKa 3.1) determined at 25 degrees C and the same ionic strength.