The rat liver ribosomal protein L6 binds to 5 S and 5.8 S rRNAs, and to initiator and elongator tRNAs. Experiments were carried out to determine if the protein has separate domains for binding each of these nucleic acids. For that purpose, nucleic acid.L6 complexes were immobilized on Sepharose and their capacity to retain 32P-labeled nucleic acids was assessed. A 5 S rRNA.L6 affinity complex binds 5.8 S [32P]rRNA indicating that L6 has separate binding sites for 5 S and 5.8 S rRNAs. A 5 S rRNA.L6 affinity complex also binds [32P]tRNAPhe, and unlabeled 5.8 S rRNA does not compete with the radioactive tRNA for binding to L6, suggesting that the ribosomal protein has a third, distinct, nucleic acid-binding domain. To determine if L6 has separate sites for the binding of elongator and initiator tRNAs, tRNAPhe. L6 and tRNAfMet.L6 affinity columns were constructed. The tRNAPhe.L6 affinity complex binds [32P]tRNAfMet, and the tRNAfMet.L6 complex binds [32P]tRNAPhe, suggesting there are distinct sites on L6 for the interaction with initiator and elongator tRNAs; however, competition experiments imply that, while there are two sites for binding tRNAs to L6, the sites do not discriminate between initiator and elongator tRNAs.