In Drosophila modifying mutations of position-effect variegation have been successfully used to genetically dissect chromatin components. The enhancer of position-effect variegation E(var)3-93D [formerly E-var(3)3] encodes proteins containing a domain common to the transcriptional regulators tramtrack and the products of the Broad complex. It interacts with a number of chromatin genes that suppress position-effect variegation. Mutations in E(var)3-93D exhibit an imprinting-like effect on the Y chromosome. This effect is transmitted paternally over several generations. Homeotic transformations in E(var)3-93D mutants indicate an involvement of the gene products in regulation of homeotic gene complexes. An antiserum raised against E(var)3-93D protein detects this chromosomal protein in a large subset of sites in polytene chromosomes. Our genetic and molecular data suggest that the proteins of E(var)3-93D are generally involved in establishing and/or maintaining an open chromatin conformation.