A bisubstrate kinetic analysis of UDPglucose:dolichylphosphate glucosyltransferase from human liver microsomes has been carried out which indicated that the kinetics follow a sequential mechanism. Inhibition studies with nucleosides, nucleotides and analogues of the substrate UDPglucose revealed that the nucleoside moiety of UDPglucose, uridine, appears to be the smallest substrate analogue that is capable of specific interaction with the enzyme at the binding site for UDPglucose. The Ki values for uridine with respect to UDPglucose were 0.17 mM or 0.1 mM for enzyme reactions at pH 5.3 or pH 7.2, respectively. Modification of the uracil moiety especially at the 6 position or lack of the 2'-hydroxyl group in the ribose moiety lessened the inhibitory potency as compared to uridine. The phosphorylated derivatives of uridine, UMP and UTP, were similar in their inhibitory properties to uridine, whereas UDP was about 10-fold more potent as an inhibitor of glucosyltransferase as compared to uridine due to product inhibition. The inhibitory properties of sugar nucleotides as substrate analogues of UDPglucose were not only dependent on the presence of the uracil moiety but were also influenced by the nature of the sugar residue. Furthermore, enzyme activity was dependent on the presence of divalent metal ions and was maximally stimulated in the presence of Ca2+.