The gene 5 protein of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication. We have generated the mutations R21A, Y26F and Y41A in the gene 5 protein and purified the mutant proteins for functional characterisation in vitro. The complex of Y26F with single-stranded DNA is disrupted at 0.8 M NaCl, the same salt concentration as that required to dissociate the native complex. However, the mutant proteins R21A and Y41A are considerably less stable and dissociate from single-stranded DNA at at 0.4 M NaCl. The fluorescence of the mutant proteins and the DNA-protein complexes they form has been compared with the wild-type protein to allow an assessment of the contribution from individual residues. We conclude that the fluorescence of Tyr-26 is 50% quenched in the complex with DNA, whereas that of Tyr-41 is fully quenched. Fluorescence titrations of the mutant proteins with poly(dT) show that all three mutant proteins can bind DNA but, in the case of Y41A, with a change of stoichiometry suggesting a loss of cooperativity. Gel retardation analysis of Y41A also shows anomalous behaviour in binding to oligonucleotides, consistent with the proposed involvement of Tyr-41 in dimer-dimer contacts in the nucleoprotein complex.