Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Pas, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development, The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity, Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun, These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals, We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val(12)]Ha-Ras, c-Raf, or c-Jun mRNA, Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP 4 signaling pathway.