Using a computer-based system for model building and analysis, three-dimensional models of 24 Drosophila melanogaster salivary gland nuclei have been constructed from optically or physically sectioned glands, allowing several generalizations about chromosome folding and packaging in these nuclei. First and most surprising, the prominent coiling of the chromosomes is strongly chiral, with right-handed gyres predominating. Second, high frequency appositions between certain loci and the nuclear envelope appear almost exclusively at positions of intercalary heterochromatin; in addition, the chromocenter is always apposed to the envelope. Third, chromosomes are invariably separated into mutually exclusive spatial domains while usually extending across the nucleus in a polarized (Rabl) orientation. Fourth, the arms of each autosome are almost always juxtaposed, but no other relative arm positions are strongly favored. Finally, despite these nonrandom structural features, each chromosome is found to fold into a wide variety of different configurations. In addition, a set of nuclei has been analyzed in which the normally aggregrated centromeric regions of the chromosomes are located far apart from one another. These nuclei have the same architectural motifs seen in normal nuclei. This implies that such characteristics as separate chromosome domains and specific chromosome-nuclear envelope contacts are largely independent of the relative placement of the different chromosomes within the nucleus.