Interactions of 5s RNA from Escherichia coli with 50s ribosomal subunit proteins L5, L18, and L25 have been evaluated by a number of criteria. From the dependence of complex formation on protein and RNA concentration in TMK buffer (50 mM Tris-HCI (pH 7.6)-20 mM MgC12-300 mM KCI), it was inferred that the three proteins differ substantially in their affinity for the nucleic acid. Measurement of the stoichiometry of association in the presence of excess protein revealed that molar protein:RNA binding ratios for L5, L18, and L25 at saturation were 0.6:1, l. l : l, and 0.7:1, respectively. The RNA molecule therefore contains no more than one specific site of attachment for each of the proteins. Solution conditions were varied to assess the effects of pH, Mg2+ concentration, and K+ concentration on the stability of the interactions. Optimal binding was observed for the L5-5s RNA complex at pH 6.5-9, [Mg2+] of 10-20 mM and [K'] of 300 to 400 mM; for the L18-5s RNA complex at pH 7.5-9. [Mg2+] of 10-20 mM and [K+] of 100-200 mM; and for the L25-5s RNA complex at pH 7.5-9, [Mg2+] of 0.3-20 mM, and [K+] of 200-300 mM. In a separate series of experiments, the association of L5 in TMK buffer was found to be cooperatively stimulated by L18 at component concentrations roughly tenfold less than were required for the association of L5 alone. The mutual influence of these two proteins upon one another was also clearly manifested in assays involving variation of pH and ionic environment. From the pattern of cooperativity, it was concluded that the binding sites for L5 and L18 in the 5s RNA are functionally related to each other, but distinct from that for protein L25.