Genetic analysis of the autogenous control of the S10 ribosomal protein operon of Escherichia coli has suggested that the secondary or tertiary structure of the leader transcript is important for this regulation. We have therefore determined the secondary structure of the leader by enzyme digestion and chemical modification. Our results suggest that the 172 base leader exists in two forms, differing only immediately upstream of the Shine-Dalgarno sequence of the first gene. We discuss the possibility that the equilibrium between these alternate structures is important for the L4-mediated regulation of translation of the S10 operon. We have also determined the structure of several mutant transcripts. Correlation of these structures with the regulatory phenotypes suggest that a hairpin about 50 bases upstream of the first gene is essential for the control of translation of the operon. Finally, our results show that a two base substitution in an eight base loop destabilizes the attached stem.