A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered β-tubulin subunit as well as wild-type β-tubulin is temperature-sensitive (ts) for growth at 40.5 °C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) . Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain β-tubulin antibody revealed that both mutant and wild-type β-tubulins were present in similar proportions in both bipolar spindles at 37 °C and multipolar spindles at 40.5 °C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant β-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 °C.