A kinase distinct from the MEK activator Raf, termed MEK kinase-1 (MEKK), was originally identified by virtue of its homology to kinases involved in yeast mating signal cascades. Like Raf, MEKK is capable of activating MEK in vitro. High-level expression of MEKK in COS-7 cells or using vaccinia virus vectors also activates MEK and MAPK, indicating that MEKK and Raf provide alternative means of activating the MAPK signalling pathway. We have derived NIH3T3 cell sublines that can be induced to express active MEKK. Here we show that induction of MEKK does not result in the activation of MAPK, but instead stimulates the stress-activated protein kinases (SAPKs) which are identical to a Jun amino-terminal kinase. We find that MEKK regulates a new signalling cascade by phosphorylating an SAPK activator, SEK1 which in turn phosphorylates and activates SAPK.