The affinity of various mutant lac DNAs for the lac repressor was examined by means of a competition binding assay. The results indicate that there exist at least two binding sites for the lac repressor. The primary binding site is coincidental with the operator, as defined by genetic studies. Mutations of the promoter or the Z gene have little or no effect on the repressor binding properties of this primary binding site, thereby confirming that the operator is distinct from lacP and Z. The secondary binding site(s), which has a 25- to 31-fold lower affinity for the repressor, is located within the operator proximal third of the Z gene.