RNA interference (RNAi) has been shown to be a powerful method to study the function of genes in vivo by silencing endogenous mRNA with double-stranded (ds) RNA. Previously, we performed in vivo RNAi screening and identified 43 Drosophila genes, including 18 novel genes required for the development of the embryonic nervous system. In the present study, 22 additional genes affecting embryonic nervous system development were found. Novel RNAi-induced phenotypes affecting nervous system development were found for 16 of the 22 genes. Seven of the genes have unknown functions. Other genes found encode transcription factors, a chromatin-remodeling protein, membrane receptors, signaling molecules, and proteins involved in cell adhesion, RNA binding, and ion transport. Human orthologs were identified for proteins encoded by 16 of the genes. The total number of dsRNAs that we have tested for an RNAi-induced phenotype affecting the embryonic nervous system, including our previous study, is 7,312, which corresponds to approximately 50% of the genes in the Drosophila genome.