Abstract : Estrogen is a key hormonal regulator of mammary epithelial cell growth. Thus, understanding the mechanisms of estrogen-mediated growth regulation is key to understanding to understanding growth disregulation in breast cancer. Current information indicates that estrogen regulates cell growth by activating the Raf-l/Mek/MAP kinase pathway and up regulating Cyclin Dl. Genetic and biochemical analysis of the Cdc37 protein has strongly implicated it in regulation of the Raf-1 Map kinase pathway, cylin/cdk association, and steroid receptor function. Here we have examined the role of p5Ocdc37 and its Hsp9O chaperone partner in Raf/Mek/MAP kinase signaling and in estrogen receptor function. We have found that p5Ocdc37 is the primary determinant of Hsp9O recruitment to Raf-1. Over expression of a p5Ocdc37 mutant which is unable to recruit Hsp9O into the Raf- 1 complex inhibited Raf-1 and MAPK activation by growth factors and estrogen. The dominant negative Cdc37 also inhibited transactivation activation of an estrogen responsive reporter gene in the human MCF-7 breast cancer cell line. We have found that co-expression of wild-type p5Ocdc3 with Raf-1 results in robust and dose-dependent activation of Raf-1 in Sf9 cells. Thus, p5Ocdc37 plays a crucial role in Raf-1 activation and MAPK pathway signaling in general and in estrogen action in particular.