The binding affinity of Escherichia coli ribosomal protein S1 for 30S ribosomal particles has been determined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one S1 protein per active 30S ribosomal subunit is approximately 2 X 10(8) M-1. The involvement of the two polynucleotide binding sites of S1 protein (site I binding single-stranded DNA or RNA, and site II binding single-stranded RNA only) in the S1--ribosomal interaction have been examined by competition experiments with polynucleotides of known affinity for the two sites. We find that site I does not contribute to the interaction; site II binding appears to provide a major part of the binding free energy, presumably by interaction of S1 with the 16S rRNA of the 30S particle. The remaining binding free energy is probably derived from the interaction of S1 protein with other proteins of the 30S subunit. The affinity of S1 for 70S ribosomes is about the same as that for the 30S subunit; the affinity of S1 for 50S subunits is much less. Binding affinities and stoichiometries of S1 protein with "inactive" 30S ribosomal subunits have also been examined.