Recent studies indicate that p50cdc37 facilitates Hsp9O-mediated biogenesis of certain protein kinases. In this report, we examined whether p50cdc37 is required for the biogenesis of the heine-regulated eIF2α kinase (HRI) in reticulocyte lysate. p50cdc37 interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50cdc37 stimulated HRI's activation in response to heine deficiency, but did not activate HRI per se. p50cdc37 function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp9O and p50cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50cdc37 with Hsp90. A C-terminal truncated mutant of p50cdc37 inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50cdc37 were detected in cultured K562 erythroleukemia cells. These results suggest that p50cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.