The Rho GTPases are molecular switches principally known for their pivotal role in regulating the actin cytoskeleton throughout the phylogeny of eukaryotes. One major function for RhoA is regulation of the actin-myosin contractile apparatus in developing epithelia. Epithelial morphogenesis in Drosophila imaginal discs is regulated by RhoA signaling and the steroid hormone ecdysone. In order to learn more about the connection between ecdysone signaling and actin cytoskeletal dynamics during epithelial morphogenesis I have characterized mutations in 5 genes [designated en(zip)] that interact genetically with mutations in the Drosophila genes encoding myosin, RhoA, and an ecdysone-induced type II transmembrane serine protease required for epithelial morphogenesis. Two of the en(zip) mutations have been previously identified as alleles of RhoA and DRhoGEF2, two members of the RhoA signaling pathway. I have employed genetic complementation assays to characterize the three unidentified en(zip) genes and narrowed the putative location of two of these genes (18-5 and 31-6) to two small genomic regions. To further characterize the en(zip) mutants I have also determined the developmental lethal phase for each homozygous mutant. Homozygous RhoA 12 - 6 and DRhoGEF212 - 3 animals die during embryogenesis. In contrast, the en(zip) mutants 12-5, 31-6, and 18-5 are pupal lethals, suggesting that the primary role of their gene products may be to regulate epithelial morphogenesis during pupal development.