The role of glucose in ajmalicine production by Catharanthus roseus was investigated in the second stage of a two-stage batch process. Activities of tryptophan decarboxylase (TDC) and anthranilate synthase (AS), two enzymes in the pathway leading to ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific ajmalicine production rate, q(P), was investigated in seven (fed-)batch cultures with constant glucose concentrations : 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the q(P) was 2.7-times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and q(P). Differences in enzyme induction and ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch.