Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentrations of 15% and 85% of air saturation, with respect to alkaloid formation and related enzyme activities. Oxygen had an evident effect on ajmalicine production : in the high DO culture production was more than 5 times higher than in the low DO culture. The differences in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not be restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active.