Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50Cdc37, the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50Cdc37 in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50Cdc37 rescues the catalytic activity of tsHck499F at 33°C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39°C). Hsp90 function is required for tsHck499F activity and its stabilization by p50Cdc37, but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50Cdc37 promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50Cdc37 might be the rate-limiting step in the association of tsHck499F with Hsp90. A truncation mutant of p50Cdc37 that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50Cdc37 may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.