lnteraction of various guanine and related nucleotides with ribonuclease Fl was studied by means of difference absorption spectra. Spectrophotometric titrations were carried out on a total of 16 nucleotides which were classined into three groups; (A)guanine and hypoxanthine nucleotides(6)，(B)8-substituted guanine nucleotides (6) and(C) dinucleotides (4)(the numbers of nucleotides are shown in parentheses). Computer-aided data processing enabled determination of the difference molar absorbance coefficient △Emax and the dissociation constant of the ribonuclease F1-nucleotide complex Kd for each nucleotide. Among group A nucleotides，guanosine 2'-phosphate showed the lowest Kd (the highest affinity) and inosine 2'-phosphate the highest Kd (the lowest affinity).The difference between their binding free energy changes was estimated to reach 2.3 kcal/mol. Among group B nucleotides，8-aminoguanosine 2'-phosphate exhibited the lowest Kd，which was even lower than that ofguanosine 2'-phosphate. The dinerence in their binding free energy changes was l.0 kcal/mol. Group C nucleotides had higher Kd than their common component，guanosine 3'-phosphate，suggesting destabilizing effect on complex formation by an adjacent nucleotide. A11 these results were similar but not completely the same for those reported on ribonuclease TI.