P58(IPK), a member of the tetratricopeptide repeat and J-domain protein families, was first recognized for its ability to inhibit the double-stranded RNA-activated protein kinase, PKR. PKR is part of the interferon-induced host defense against viral infection, and down-regulates translation initiation via phosphorylation of eukaryotic initiation factor 2 on the alpha-subunit. P58(IPK) is activated in response to infection by influenza virus, and inhibits PKR through direct protein-protein interaction. Previously, we demonstrated that the molecular chaperone heat shock protein 40 (hsp40) was a negative regulator of P58(IPK). We could now report that influenza virus activates the P58(IPK) pathway by promoting the dissociation of hsp40 from P58(IPK) during infection. We also found that the P58(IPK)-hsp40 association was disrupted during recovery from heat shock, which suggested a regulatory role for P58(IPK) in the absence of virus infection. The PKR pathway is even more complex as we show in this report that the molecular chaperone, hsp/Hsc70, was a component of a trimeric complex with hsp40 and P58(IPK). Moreover, like other J-domain proteins, P58(IPK) stimulated the ATPase activity of Hsc70. Taken together, our data suggest that P58(IPK) is a co-chaperone, possibly directing hsp/Hsc70 to refold, and thus inhibit kinase function.