Recent studies have demonstrated that Rous sarcoma virus-transformed baby hamster kidney (RS-BHK) cells express twofold higher levels of those N-linked oligosaccharides that contain the sequence [GlcNAc-beta(1,6)Man (1,6)] compared to nontransformed parental BHK cells (Pierce and Arango, J. Biol.Chem. 261, 10772 [1986]). We have investigated in RS-BHK and BHK cells the activity of UDP-GlcNAc:alpha-D-mannoside beta(1,6)N-acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS-BHK cells. We have measured GnT V activity using UDP-[3H]-GlcNAc and a synthetic oligosaccharide acceptor, GlcNAc beta(1,2)Man alpha(1,6)Man beta-O-(Ch2)8COOCH3, separating the radioactive product by a newly devised reverse-phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS-BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAc beta(1,6)Man] sequences in the RS-BHK cells. The apparent Km values of the enzymes in RS-BHK and BHK cell sonicates for UDP-GlcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V-specific activity in RS-BHK cells is not caused by the presence in these cells of a GnT V with markedly different kinetic properties.