Mixed lineage kinase‐3 (MLK‐3) is a 97 kDa serine/threonine kinase with multiple interaction domains, including a Cdc42 binding motif, but unknown function. Cdc42 and the related small GTP binding protein Rac1 can activate the SAPK/JNK and p38/RK stress‐responsive kinase cascades, suggesting that MLK‐3 may have a role in upstream regulation of these pathways. In support of this role, we demonstrate that MLK‐3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK‐3 catalyzed the phosphorylation of SEK1 in vitro, and co‐transfected MLK‐3 induced phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Furthermore, interactions between MLK‐3 and SEK and MLK‐3 and MKK6 were observed in co‐precipitation experiments. Finally, kinase‐dead mutants of MLK‐3 blocked activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK‐3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20‐like kinases.