The bacteriophage T4 alpha- and beta-glucosyltransferases (AGT and BGT) catalyse the transfer of glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine of T4 DNA in an alpha- and beta-conformation, respectively. Following the 3D structure of BGT and a secondary structure alignment of AGT and BGT, we performed a site-directed mutagenesis of AGT. A two-domain structure was deduced, with an open substrate-free and a closed substrate-bound conformation. We also identified specific amino acids involved in DNA binding. The identification of a protein-protein interaction of AGT and gp45 which is a part of the T4 replication complex supports the idea that T4 DNA is alpha-glucosylated immediately after synthesis. BGT then glucosylates those hydroxymethyl cytosines not previously served by AGT.