Transgenic flies were established in which ectopic expression of boundary element-associated factor (BEAF) 32A was targeted to the Drosophila eye imaginal disc. The eyes of the adult fly displayed a severe rough eye phenotype. When these eyes were sectioned, most ommatidia were found to be fused and irregularly shaped rhabdomeres were observed. In the developing eye imaginal disc, expression of BEAF32A inhibited differentiation of photoreceptor cells. Expression of BEAF32A also induced extensive apoptosis of eye imaginal disc cells and, consistent with this, co-expression of baculovirus P35 in the eye imaginal disc suppressed the BEAF32A-induced rough eye phenotype. To investigate the effects of BEAF32A on regulation of chromatin structure, genetic crosses of the BEAF32A-overexpressing flies with loss-of-function mutants for genes encoding other boundary element-binding factors or regulators of chromatin structure were conducted. Interestingly, half-dose reduction of the su(Hw) gene strongly enhanced the rough eye phenotype induced by BEAF32A. Furthermore, genetic crosses of the transgenic flies with loss-of-function mutants for genes interacting with Polycomb revealed specific links between BEAF32A and genes such as Distalless and kohtalo, suggesting a relation to the chromatin insulator function of BEAF. In addition, genetic crosses of transgenic flies expressing BEAF32A with a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the BEAF32A-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify targets of BEAF32A and its positive or negative regulators in Drosophila.