Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E. coli 16 S rRNA. This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair. Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively. We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding. Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7. However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A. Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding. Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7. S7 binding was also affected by mutations in helix 29. Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect. Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B.