Transcription of Escherichia coli biotin operon is repressed by biotin repressor in the presence of biotinyl-5'-adenylate as a corepressor. To determine precisely the site of action of biotin repressor on the operator sequence, DNaseI footprinting experiments were performed on the PCR-produced biotin operator and its mutants. The results indicate that the repressor binds to the wild-type operator as well as mutated operator sequence at +15 position or -15 position, and protects the 39-base region from nucleotide -19 to +20 of the upper strand, and the 40-base region from nucleotide -22 to +18 of the lower strand, with a few hypersensitive sites. This is consistent with previous speculation that the biotin operator is an approximately 40 bp imperfect palindromic DNA sequence capable of binding with two molecules of biotin repressor. However, the protection pattern of the mutant operator which lacks half of the palindromic structure is quite different from the corresponding region of the wild type. Though two repressor monomers bound to the mutant operator, as revealed by parallel binding studies [Lin, Shiuan, and Campbell (1991) Biochim. Biophys. Acta 1090, 317-325], only 12 to 13 bp on the DNA sequence was protected, suggesting that one monomer of the repressor dimer is hanging near the DNA backbone of the mutant operator even though the biotin repressor is functioning as a dimer.