In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.