The c-Raf-1 proto-oncoprotein is a Ras-GTP-regulated protein kinase that associates in situ with 14-3-3 proteins, which are naturally dimeric. In COS cells, recombinant Raf is found in oligomeric assemblies. To examine whether induced oligomerization can alter Raf kinase activity, sequences encoding the FK506-binding protein FKBP12 were fused to the amino terminus of c-Raf-1, introducing a binding site for FK506. Oligomerization of recombinant FKBP-Raf in situ, induced by the addition of the dimeric FK506 derivative FK1012A, activated Raf kinase activity at least half as well as epidermal growth factor (EGF). As with EGF, activation of FKBP-Raf by FK1012A is entirely Ras-GTP dependent. Thus oligomerization of Raf per se promotes Raf activation through a Ras-dependent mechanism.