rough (ro) encodes a homeobox transcription factor required for proper specification of photoreceptor cells R2 and R5 in Drosophila eye development. To identify the transcriptional targets through which ro acts to specify the R2/R5 neuronal sub-type, we screened enhancer trap lines expressed in developing photoreceptors for those whose expression patterns were altered when ro function was inactivated. In this way we identified two potential ro targets, which are also targets of the zinc finger transcription factor glass (gl). We also identified an enhancer trap line that exhibits altered morphogenetic furrow expression in a ro mutant background. Finally, we have molecularly characterized an enhancer trap line, AE33, that was identified in earlier screens as a target of both ro and gl (freeman et al., 1992; Treisman and Rubin, 1996). The transcript interrupted by AE33 shares similarity with the mammalian vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases that is associated with actin filaments, focal adhesions, and dynamic membrane regions (Haffner et al., 1995) with enabled (ena), a substrate of the Drosophila Abl tyrosine kinase (Gertler et al. 1995) and with two human Expressed Sequence Tags (ESTs).