ABSTRACT This article reports the identification and characterization of a DBL‐like guanine nucleotide exchange factor (GEF) in Drosophila , called GEFmeso, as a novel binding target of the Ras‐like GTPase Ral. Previous studies suggested that some aspects of Ral activity, which is involved in multiple cellular processes, are mediated through regulation of Rho GTPases. Here we show in vitro association of GEFmeso with the GTP‐bound active form of Ral and the nucleotide‐free form of the Rho GTPase Cdc42. GEFmeso fails to bind to other Rho GTPases, showing that Cdc42 is a specific interaction partner of this GEF. Unlike Ral and Cdc42, which are ubiquitously expressed, GEFmeso exerts distinct spatio‐temporal expression patterns during embryonic development, suggesting a tissue‐restricted function of the GEF in vivo. Based on previous observations that mutations in Cdc42 or overexpression of mutant alleles of Cdc42 lead to distinct effects on wing development, the effects of overexpression of dominant‐negative and activated versions of Ral on wing development were analyzed. In addition, GEFmeso overexpression studies as well as RNAi experiments were performed. The results suggest that Ral, GEFmeso and Cdc42 act in the same developmental pathway and that GEFmeso mediates activation of Cdc42 in response to activated Ral in the context of Drosophila wing development.‐Blanke, S., Jäckle, H. Novel guanine nucleotide exchange factor GEFmeso of Drosophila melanogaster interacts with Ral and Rho GTPase Cdc42. FASEB J. 20, 683–691 (2005)