atonal (ato) encodes a basic helix-loop-helix protein and is required for the specification of R8 photoreceptor cells in Drosophila. In the eye imaginal discs, expression of Ato protein is initially in a dorsoventral stripe of cells anterior to the morphogenetic furrow (MF). In the MF, this stripe expression is resolved into regularly spaced clusters of Ato-positive cells, the proneural clusters, which are intervened with Ato-negative cells. Another basic helix-loop-helix protein, Daughterless (Da), dimerizes with Ato and is expressed at an enhanced level in Ato-expressing cells. Here we show that during the late stages of proneural clusters, the mitogen-activated protein kinase (MAPK) is activated in proneural clusters. Normal ato or da activity is required for maintenance of MAPK activation. Furthermore, in ato or da mutants, Ato expression is expanded to all cells in the MF, suggesting that ato and da are required for Ato repression in cells between proneural clusters. By changing the MAPK activity in proneural clusters, we show that MAPK activation mediates Ato repression nonautonomously. Consistently, hyperactivation of the MAPK in a stripe of cells posterior to or overlapping the Ato stripe eliminates the formation of proneural clusters. Taken together, these results suggest that a negative regulatory loop involving MAPK activation and Ato repression is required for the generation of evenly spaced proneural clusters.