The cysteine proteinase I gene of Dictyostelium discoideum is a developmentally regulated single copy gene. Specific sites in the 5' and the 3' flanking regions of the gene were cleaved by an endogenous nuclease when the gene was being transcribed. The majority of these sites were not cut when the gene was inactive. A dramatic change in the pattern of micrococcal nuclease and DNase I hypersensitive sites occurred in the 5' flanking region when transcription commenced at the 8 h stage of development. The major sites, doublets at -220/-300 bp and -670/-770 bp upstream of the transcription start site, corresponded to those cut by the endogenous nuclease. When transcription subsequently ceased the hypersensitive sites did not significantly change, indicating the gene remained in an activated state. The micrococcal nuclease hypersensitive sites in the 3' flanking region did not change significantly during development.