The position of acidic, basic and phenolic amino acid residues of nucleoproteins and proteins of the TMV strains vulgare (and mutants of vulgare), dahlemense, and U 2 were determined by comparative potentiometric and spectrophotometric titrations in order to obtain more precise information about the interactions of proteins. In the nucleoproteins of all these mutants only 8 carboxylgroups of 16 (vulgare, A 14, Ni 1927 and U 2) and 8 of 15 (flavum, Ni 116, Ni 109 and Ni 606) can be titrated. Thus it is concluded that only these 8 groups are situated near the surface. The differences in the titration curves, measured at I=0.1 and I=0.02 show that one amino acid with pK 5.2 is titrated in vulgare but not flavum, Ni 116 and Ni 606. The titration curves of the mutant proteins differ from one another and from that of vulgare between pH 7 and 6 and this is probably due to aggregation which starts for each protein at different pH values. In addition, the differences in titration curves at pH values 4.5 are probably due structural changes. Also the titration curves of the strain proteins of vulgare, dahlemense and U 2 are different. The first binding of protons during the titration of the proteins occurs before pH 7 for U 2, about pH 7 for vulgare, and at pH 6.6 for dahlemense. Opalescence and sedimentation values indicate that aggregation takes place in the same sequence. At pH values >5 vulgare protein binds more protons than dahlemense and fewer than U 2. Due to the isoelectric state the U 2 protein precipitates at pH 5.5 (nucleoprotein at pH 4.1), dahlemense protein does not precipitate (although nucleoprotein does precipitate at pH 3.15) and the vulgare nucleoprotein and protein precipitate at pH 3.9. Spectrophotometric titrations indicate that two of 4 tyrosine residues are ionized in vulgare and mutant nucleoproteins (except Ni 2068 where 1.2 of 3 tyrosine residues are ionized). The dissociation of the first proton of a tyrosine residue begins with the depolymerization of the nucleoprotein which is not completed until the titration end point is reached (pH 12.6). The ionization of the tyrosine residues begins at lower pH values in proteins than in nucleoproteins and, furthermore, the protein contains an additional ionized tyrosine residue. In the nucleoprotein and protein of U 2 4.2 of 6 tyrosine residues are titrated whereas in dahlemense 2.3 and 3.8 tyrosine residues are titrated in nucleoprotein and protein, respectively. Binding studies with labelled lead ions show that vulgare, related mutants, and U 2 bind two lead ions per subunit whereas dahlemense binds 2.5. The comparison of all results shows that a constant number of acidic amino acid residues must be on the surface of the TMV particle in order to obtain a stable TMV nucleoprotein. Furthermore, two intermolecular interactions between carboxyl-carboxylate groups must be present for the aggregation of proteins to occur.