The homeobox gene sine oculis (so) is required for the development of the entire visual system in Drosophila, which includes the compound eyes, the ocelli, the optic lobe of the brain and the Bolwig's organ. During ocelli development, so expression labels, together with eyes absent (eya), the emergence of the ocellar precursor cells in the third instar eye-antennal disc. Footprinting and misexpression studies have led to the proposal that the Pax6 homologue twin of eyeless (toy) directly regulates the initiation of so expression in ocellar precursor cells. However, so expression in a toy loss-of-function mutant background has not been yet analyzed due to the lack of eye-antennal disc development in strong toy mutant alleles. Using an embryonic eye primordium-specific enhancer of toy, we have rescued the developmental defect of a strong toy mutant allele and analyzed so expression in the ocelli primordium of toy loss-of-function eye-antennal discs during third instar larva. The results show that so expression is only marginally affected in the absence of Toy transcriptional activity and that the toy positive effect on so expression is largely eya-mediated. These results suggest that eya is the main factor controlling both initiation and maintenance of so expression in ocellar precursor cells. In addition, we present the characterization of a new minimal eye/ocellus-specific enhancer of the so gene.