Many previous studies have been directed toward obtaining a physical visualization of the relationship between the protein and RNA in the ribosomal subunits isolated from Escherichia coli. The current study is the first report where an attempt has been made to directly assess interactions between a pair of isolated ribosomal proteins separate from the intact system by means of sedimentation equilibrium analysis. The molecular weights of the proteins S3, S4, S5, S6, S7, S8, and S20 from the 30S subunit of the E. coli ribosome were determined under conditions of assembly of the subunit by sedimentation equilibrium. All of the proteins exhibited molecular weights consistent with monomeric behavior (i.e., in agreement with the measurement of the ultimate molecular weight in denaturing solvents as reported in other studies as well as in the current study) except S8 which indicates a tendency to self-associate. Hydrodynamic measurements on the proteins indicate that these proteins are not completely disorganized in solution such as a random coil, although not as compact as globular proteins. The frictional coefficient ratios found for these ribosomal proteins range from 1.4 to 1.9. The hydrodynamic data are discussed as containing some evidence that stable interaction sites could exist in the proteins. The molecular weight data are considered pertinent to a sedimentation equilibrium study of protein-protein interactions that may be occurring in the ribosomal subunits. Two proteins, S3 and S5, considered in this investigation were found to exhibit no tendency to self-associate under conditions of reassembly. When the two proteins are mixed under those same conditions, however, a species with a molecular weight greater than that of either S3 or S5 is observed to be formed. The interpretation is presented that a molecular interaction between S3 and S5 is the cause. The system is described as containing S3, S5, and a complex between S3 and S5 with a stoichiometry of 1:1 and an association equilibrium constant of 5.7 times 10-5 l./mol (delta G-o equals minus 7.25 kcal/mol). Since the association appears to be specific and of moderate strength, it is concluded that the interaction could have some pertinence with respect to conferring a structural arrangement in the ribosomal subunit. Moreover, it is concluded that protein-protein interactions, in general, must be considered in addition to the well documented significant RNA-protein relationships when models for ribosome structure and assembly are formulated.