A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892-5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non-cooperative response observed in the case of IMP. It is shown that the 5'-phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, delta G, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences delta G (analog) - delta G (AMP) = delta G obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule. It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6-phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5,5'-dithio-bis(2-nitrobenzoic acid) at 4 degrees C; (c) association of two molecules of dimer is strengthened at 4 degrees C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6-phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5'-dithio-bis(2-nitrobenzoic acid) by the binding of the analog at 4 degrees C; (c) the analog dissociates the low amount of tetramer which is present at 4 degrees C in the absence of AMP into two molecules of dimer. These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.