The highly conserved nature and tissue specificity of the seven vertebrate beta-tubulin isotypes provide circumstantial evidence that functional differences among isotypes may exist in vivo. Compelling evidence from studies of bovine brain beta-isotypes indicated significant conformational and functional differences in vitro and implied that these differences could be related to in vivo function. A previously uninvestigated parameter of potential importance in assessing functional significance is molecular stability. We examined the relative stability of alphabetaII and alphabetaIII tubulin dimers purified from bovine brain. The use of probes to monitor the exposure of hydrophobic areas and sulfhydryls and the loss of colchicine binding, all of which are known to accompany tubulin's time-dependent loss of function, showed an acceleration of these criteria in alphabetaII relative to alphabetaIII when the isotypes were incubated at 37 degrees C. Studies using differential scanning calorimetry suggested that unfolding of the isotypes at approximately 60 degrees C and decay at 0 degrees C were both highly cooperative. It was also observed that alphabetaIII had a higher melting temperature and a larger population of molecules retaining tertiary structure after incubation at 0 degrees C for 20 h. These studies support the conclusion that alphabetaIII is significantly more stable than alphabetaII and raise the possibility that differences in relative stabilities of tubulin isotypes may be important in regulating the functional properties of microtubules in vivo.