In the evolutionarily distant yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, genetic evidence suggests that activation of pheromone-induced mitogen-activated protein kinase (MAPK) cascades involves the function of the p21cdc42/racl-activated protein kinases (PAKs) Ste20 and Shk1, respectively. In this report, we show that purified Ste20 and Shk1 were each capable of inducing p42MAPK activation in cell-free extracts of Xenopus laevis oocytes, while a mammalian Ste20/Shk1-related protein kinase, p65pak (Pak1), did not induce activation of p42MAPK. In contrast to p42MAPK, activation of JNK/SAPK in Xenopus oocyte extracts was induced by both the yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. Our results demonstrate that MAPK cascades that are responsive to PAKs are conserved in higher eukaryotes and suggest that distinct PAKs may regulate distinct MAPK modules.