Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.