Yeast ribosomal protein S14 (rpS14) binds to two different RNA molecules: (1). helix 23 of 18S rRNA during its assembly into 40S ribosomal subunits and (2). a stem-loop structure in RPS14B pre-mRNA to repress expression of the RPS14B gene. We used the three-dimensional structure of Thermus thermophilus ribosomal protein S11, a bacterial homologue of rpS14, as a guide to identify conserved, surface-exposed amino acid residues that are likely to contact RNA. Eight residues that met these criteria were mutated to alanine. Most of these mutations affected interaction of rpS14 with either helix 23 or the RPS14B stem-loop RNA or both. Assembly of 40S ribosomal subunits and repression of RPS14B were also affected. S11 contains an extended carboxy-terminal domain rich in basic amino acids, which interacts with rRNA. We systematically evaluated the importance of each of the last ten amino acid residues in the basic, carboxy-terminal tail of yeast rpS14 for binding to RNA, by mutating each to alanine. Mutations in nine of these residues decreased binding of rpS14 to one or both of its RNA ligands. In addition, we examined the importance of four structural motifs in helix 23 of 18S rRNA for binding to rpS14. Mutations that altered either the terminal loop, the G-U base-pair closing the terminal loop, or the internal loop affected binding of rpS14 to helix 23.