Mitogen-activated protein (MAP) kinases orchestrate the effects of many extracellular stimuli on cells. The serine/threonine protein kinase MEKK1 is an upstream activator of the MAP kinases c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), extracellular signal-regulated kinase (ERK), and p38 as well as NF-B. In a yeast two-hybrid interaction screen to identify proteins that bind to an N-terminal fragment of MEKK1 (amino acids 1-719), the actin-crosslinking protein โฃ-actinin was identified as a MEKK1-binding protein. Over-expressed MEKK1 co-immunoprecipitated with โฃ-actinin in cell lysates. Both endogenous and over-expressed MEKK1 colocalized with โฃ-actinin along actin stress fibers and at focal adhesions. Residues 221-559 of MEKK1 bound to purified โฃ-actinin in vitro, indicating that the interaction is direct, and this fragment localized to actin filaments in cells. MEKK1 kinase activity was not required for association with actin filaments, because a catalytically inactive mutant of MEKK1 (MEKK1 D1369A) localized to stress fibers. These results provide strong evidence for the interaction between MEKK1 and โฃ-actinin. Thus, restriction of the kinase to the actin cytoskeleton may serve to regulate its specificity towards downstream targets.