To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We mobilized the Gene Search (GS) P element and identified 42 lines with insertions in unique loci, including leak/roundabout2, which encodes an axon guidance receptor and confirms the utility of our screen. The genes we identified encode proteins of diverse classes, some acting near the cell surface and others in the cytoplasm or nucleus. We found that one GS line drove misexpression of the NF-kB transcription factor Dorsal, causing motor axons to bypass their correct termination sites. In the developing visual system, Dorsal misexpression also caused photoreceptor axons to reach incorrect positions within the optic lobe. This mistargeting occurred without observable changes of cell fate and correlated with localization of ectopic Dorsal in distal axons. We found that Dorsal and its inhibitor Cactus are expressed in photoreceptors, though neither was required for axon targeting. However, mutation analyses of genes known to act upstream of Dorsal revealed a requirement for the interleukin receptor-associated kinase family kinase Pelle for layerspecific targeting of photoreceptor axons, validating our screen as a means to identify new molecular determinants of nervous system development in vivo. N ERVOUS system function relies upon patterned development of neurons and controlled establishment of synaptic connections. Axons of developing neurons are guided by instructive cues to their appropriate target areas, where they then select their correct synaptic partners. Understanding how axons are guided by these cues and how they distinguish appropriate targets from inappropriate ones is a central issue in developmental neurobiology. Drosophila melanogaster has proven a successful model with which to apply genetics to this issue and study fundamental and evolutionarily conserved molecular mechanisms that underlie axon guidance. Both forward and reverse genetics approaches have been used to identify mutants with disrupted patterning of axon tracts in either the embryonic ventral nerve cord (VNC) or the developing visual system. Despite considerable successes in identifying novel genes required for axon guidance and targeting, forward genetic screening approaches can be limited by technical challenges and by genetic redundancies which obscure the detection of mutant phenotypes, undoubtedly leaving many genes and molecular pathways remaining to be described. We have exploited P-element transposition in Drosophila, combined with the GAL4-UAS gene misexpression system, to identify genes that can influence axon guidance and reveal genes that may not emerge readily from forward genetic screens. We have mobilized the Gene Search (GS) P element, which supports GAL4-directed expression of genes that flank the site of insertion (Toba et al. 1999), and screened for genes whose misexpression can influence the stereotypic patterning of emb...