sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.