We have isolated part of the glnA region of Escherichia coli K-12 as a 6.4 Md DNA fragment on the ColE1 hybrid plasmid pACR1. DNA fragments from pACR1 obtained by cleavage with certain restriction endonucleases were subcloned into the pBR322 cloning vehicle. Recognition sites for the endonucleases BamHI, SmaI, BglII, and EcoRI were localized inside the glnA gene sequence. De novo synthesized polypeptides, employing minicells that carried some of these plasmids, allowed us to determine the direction of transcription of the glnA gene relative to an adjoining gene that codes for a 65 000 dalton protein.