Cardiotonic glycosides, compounds of great interest due to their pharmacological applications, are produced by Digitalis genus plants. Several steps are involved in the synthesis of these compounds which are catalyzed by specific enzymes whose activity may control the cardenolide production. One of these enzymes is Cyt P450 scc, responsible for the cholesterol side-chain cleavage. The activity of this enzyme is regulated by factors such as light, xenobiotics and ionic equilibrium. There are several studies about the effect of the above mentioned factors on growth and cardenolide production in vitro tissue cultures of Digitalis plants. It has been shown that white and red light enhance the growth of Digitalis callus tissues, whereas blue light inhibits biomass production although it increases cardenolide accumulation. Among the xenobiotic compounds, methylcholantrene has no effect on biomass or cardenolide production; in contrast the effect of phenobarbital on growth and cardiotonic glucoside production of Digitalis callus tissues is very clear and is dependent on the xenobiotic concentration in the culture medium. The presence of phenobarbital (80 mg/l) slightly inhibits growth and cardenolide biosynthesis in Digitalis callus culture although at the same time clearly reduces its degradation. In contrast, callus tissues cultured in the presence of a phenobarbital concentration of 40 mg/l reach a similar biomass to those grown without the addition of the xenobiotic compound. However cardenolide production of callus tissues is clearly increased through the action of phenobarbital on the number of mitochondria and on the activity of Cyt P450 scc. From ultrastructural studies of callus the production of Digitalis callus tissues; it can be inferred that a substructural differentiation is needed for cardenolide biosynthesis. The effect of an ionic imbalance on the digoxin cotent in Digitalis lanata plants has also been studied.