The 68C intermolt puff of Drosophila melanogaster contains a cluster of three glue protein genes, Sgs-3, Sgs-7, and Sgs-8. Analyses of chromosomal rearrangments which break near the glue gene cluster show that a region of no more than 20 kilobase-pairs (kb) is required for expression of the genes and for formation of the 68C puff. This result is supported by P-element-mediated-transformation experiments in which defined segments of the 68C region are introduced back into the fly genome. Based on the criteria of correct tissue- and stage-specific expression, transcription of an RNA of appropriate size and abundance, and production of an sgs-3 protein, the correctly regulated expression of the Sgs-3 gene requires less than 3.4 kb of total flanking sequences, approximately 2.3 kb 5' and 1.1 kb 3'. When upstream sequences are truncated at 130 base-pairs, low levels of Sgs-3 expression are observed in some cases, with normal tissue- and stage-specific expression retained. Formation of a new intermolt puff at the site of insertion is observed for transformants in which the introduced DNA contains all three 68C glue genes, but not for those which contain only an introduced Sgs-3 gene, even for cases in which the gene is abundantly expressed. An attempt was made to recover lethal mutations in genes closely flanking the 68C glue protein genes by screening mutagenized chromosomes over large deficiencies which delete the 68AC region. Although a large number of lethal and semi-lethal mutations were recovered, including many which define new complementation groups, none maps close to the region of the 68C glue gene cluster.