Genetic screens in Drosophila have identified p50cdc37 to be an essential component of the sevenless receptor/MAP kinase signaling pathway, but the both the function and target of p50cdc37 in this pathway has not been defined.  Here we have examined the role of p50cdc37 and its Hsp90 chaperone partner in Raf/Mek/MAP kinase signaling biochemically.   We have found that coexpression of wild-type p50cdc37 with Raf-1 results in robust and dose-dependent activation of Raf-1 in Sf9 cells.  In addition, p50cdc37 greatly potentiated v-Src-mediated Raf-1 activation.  Moreover, we have found that p50cdc37 is the primary determinant of Hsp90 recruitment to Raf-1.  Overexpression of a p50cdc37 mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors.  Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50cdc37/Hsp90 heterodimer and Raf-1 kinase activation by serum.  Activation of Raf-1 via baculoviral coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50cdc37 or by GA.  Thus, formation of a ternary Raf-1/p50cdc37/Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling.   These results provide the first biochemical evidence for the requirement of the p50cdc37/Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.