Pyruvate kinase (EC 2.7.1.40) from skeletal muscle (designated MI-type) is commonly believed not to be regulated in uiuo either by interaction with regulatory ligands or by protein phosphorylation-dephosphorylation. This apparent lack of regulatory interactions has been viewed as consistent with the proposed rble of the enzyme in intermediary metabolism in that there is no gluconeogenesis in skeletal muscle. The proposed phosphofructokinase-fructose 1,6-bisphosphatase substrate cycle (Newsholme & Crabtree, 1970) and the pathway of alanine formation in skeletal muscle as proposed by Goldstein & Newsholme (1976) and Snell & Duff (1977) make it necessary to question whether M,-type pyruvate kinase might not have an important regulatory function(s) in skeletal muscle. Accordingly, the regulatory properties of pyruvate kinase isolated from rabbit skeletal muscle were re-investigated. In agreement with Carminatti et al. (1971), the skeletalmuscle enzyme was shown to be inhibited in an allosteric manner by phenylalanine at alkaline pH. M I;type pyruvate kinase has an obligate requirement for two bivalent cations (Mez+ or Mn2+) per active site (Gupta et al., 1976). The Mgz+-dependency of phenylalanine inhibition was accordingly studied. Inhibition and the degree of co-operativity in phenylalanine interaction with pyruvate kinase were markedly Mg2+dependent. The apparent K, and Hill coetlicient, h, increased with decreasing Me2+ concentration. Inhibition was competitive with respect to phosphoenolpyruvate, but, whereas binding of phenylalanine to the enzyme showed co-operativity, the plot of u versus [phosphoenolpyruvate] complied to the MichaelisMenten equation irrespective of whether phenylalanine was present or absent. There is no question of the observed Me2+-dependency of phenylalanine inhibition being explained in terms of Me2+ chelation by phenylalanine. ATP altered markedly the kinetics of phenylalanine inhibition. At low total Me2+ concentrations (e.g. 1.OmM) at pH7.4-8.4, plots of u versus [phosphoenolpyruvatel were sigmoidal in the presence of ATP ( 5 m ~ ) and phenylalanine. Sigmoidicity was not apparent unless both inhibitors were present together. The first question one must ask is whether the apparent sigmoidicity is simply a function of Mg2+ chelation by ATP. EDTA at concentrations sufficient to produce significant chelation of available Me2+ did not interact with phenylalanine to produce any significant sigmoidicity in plots of u versus [phosphoenolpyruvatel. Thus the specificity of the inhibition with respect to ATP argues against non-specific chelation as a basis for the apparent allosterism. An alternative approach was to use the computer program devised by Macfarlane et al. (1974), which makes it possible to vary the concentration of free (uncomplexed) Me2+ (Mgge) while maintaining constant the concentrations of complexed species (e.g. MgATP2-). By adopting this approach (Fig. l), it was shown, for example, that at pH 8.0 and 0.2 mM-Mgk inhibition by phenylalanine (1.25 mM) in the presence of ATP (5 mM) was sigmoidal (h 1.6). N o co-operativity was apparent, however, at M g k concentrations of 0.5 mM or above. Notwithstanding, an apparent K, of 0.15mM for phenylalanine was determined at p H 8.0 and 0.5 mM-Mgk. Whereas the mechanism of coupled ATP and phenylalanine inhibition remains to be elucidated, it is clear that the interaction might have a regulatory function in uiuo. Co-operativity admittedly is increased by high pH and low Mg& concentrations. Nonetheless, this type of inhibition may be relevant in, for example, the phase of increased skeletal-muscle protein