Eukaryotic genomes function in the context of chromatin, but the roles of most nonhistone chromosomal proteins are far from understood. The D1 protein of Drosophila is an example of a chromosomal protein that has been fairly well characterized biochemically, but has nevertheless eluded functional description. To this end, we have undertaken a gain-of-function genetical analysis of D1, utilizing the GAL4-UAS system. We determined that ubiquitous overexpression of D1 using the Act5C- or tubP-GAL4 drivers was lethal to the organism during larval growth. We also ectopically expressed D1 in a tissue-limited manner using other GAL4 drivers. In general, ectopic D1 was observed to inhibit differentiation and/or development. We observed effects on pattern formation of the adult eye, bristle morphogenesis, and spermatogenesis. These phenotypes may be the consequence of misregulation of D1 target genes. A surprising result was obtained when D1 was overexpressed in the third instar salivary gland. The polytene chromosomes exhibited numerous ectopic associations such that spreading of the chromosome arms was precluded. We mapped the sites of ectopic pairing along the polytene chromosome arms, and found a correlation with sites of intercalary heterochromatin. We speculate that these sites comprise the natural targets of D1 protein activity and that D1 is involved in the ectopic pairing observed for wild-type chromosomes. Together, our data suggest that D1 may influence multiple biochemical activities within the nucleus.