We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.