The Drosophila homeo box gene rough is required in photoreceptor cells R2 and R5 for normal eye development. We show here that protein expression is limited to a subset of cells in the developing retina where is transiently expressed for 30-60 hr. The rough protein is first expressed broadly in the morphogenetic furrow but it is rapidly restricted to the R2, R3, R4, and R5 precursor cells. Ubiquitous expression of rough under the control of the hsp 70 promoter in third-instar larvae suppresses the initial steps of ommatidial assembly. Structures derived from other imaginal discs are not affected. Ectopic expression of rough in the R7 precursor, through the use of the sevenless promoter, causes this cell to develop into an R1-6 photoreceptor subtype; however, this cell still requires sevenless function for its neural differentiation. Taken together with previous analysis of the rough mutant phenotype, these results suggest that the normal role of rough is to establish the unique cell identity of photoreceptors R2 and R5.