MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator.