The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes. To elucidate the function of the yeast cytosolic hsp70 Ssa1p in vivo, we characterized a Saccharomyces cerevisiae ssa1 temperature-sensitive mutant (ssa1-134). After shifting to the restrictive temperature (37 degreesC), ssa1-134 mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2 N DNA content. We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed. Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJ homologue in yeast) was much weaker compared with wild-type Ssa1p. These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase. In support of this idea, a ydj1 null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than ssa1-134. Furthermore, both ssa1-134 and ydj1 null mutant cells exhibited greater sensitivity to anti-microtubule drugs. Finally, the observation that SSA1 and YDJ1 interact genetically with a gamma-tubulin, TUB4, supports the idea that they play a role in the regulation of microtubule formation.