Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60(v-src) in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60(v-scr)-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60(v-scr) than the parental wild-type strain at 23 degrees C. However, p60(v-scr) levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G(1) and G(2)/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G(2)/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60 sensitive and produces wild-type amounts of p60(v-scr) at permissive temperatures but p60(v-scr) becomes p60 -resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60(v-scr)-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60(v-scr) levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60(v-scr) in a soluble, biologically active form.