A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.