Involvement of the Embden-Meyerhof Parnas and the pentose phosphate pathways in glucose oxidation in glucose oxidation in tobacco callus was examined. Marked changes in the activities of glucokinase, aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase were observed during culture of tobacco callus under shoot-forming and non-shoot-forming conditions. Activities of these enzymes were higher in shoot-forming tissue than in non-shoot-forming tissue. Furthermore, the activities of the pentose phosphate pathway enzymes showed greater differences than those of the Embden-Meyerhof-Parnas pathway. Confirmation of these findings was obtained by investigating the contributions of 14C from [14C-1]- and [14C−6]-glucose to CO2 released. The significance of these findings on glucose oxidation in relation to the shoot-initiation process are discussed.