Previous experiments have identified a region that is required for the expression of the Drosophila glue protein gene Sgs-4 and is located 300-500 base pairs upstream from the structural gene. The chromatin in this region changes conformation and becomes hypersensitive to DNase I digestion when the gene becomes active, a change that apparently induces additional conformational changes near the site of transcription initiation. To learn more about the DNA sequence requirements for the function of this region, we analyzed three naturally occurring Sgs-4 under-producers. In two of these strains, a single base pair change within the hypersensitive region is correlated with a 50% reduction in the amount of Sgs-4 RNA produced. Another strain, which has multiple 5' lesions, is severely reduced in Sgs-4 expression and in the DNase hypersensitivity of the upstream region. Several of the sequence changes in this extreme underproducer lie near hypersensitive sites, suggesting that they inhibit the appearance of the normal DNase hypersensitive conformation.