Plant cell suspension cultures of Nicotiana tabacum cv. Wisconsin-38 accumulate nornicotine as the major alkaloid, unlike the parent plant where nicotine predominates. Nicotine was added to cultures of this cultivar, grown with differing degrees of dependence on photosynthesis for the provision of organic constituents, and also to cultures grown on medium that was optimised for alkaloid accumulation. Under optimal conditions nicotine was converted to nornicotine at an efficiency close to 100%, over 10 d. Conversion appeared to be intracellular, with nicotine being taken up from the medium, demethylated to nornicotine and the product released. Illumination of cultures caused a slight decrease in conversion but, when cultures were partially photosynthetic, both growth and the degree of biotransformation were decreased by approximately 40%. As well as the four main tobacco alkaloids several minor alkaloids were detected in cultures after incubation with added nicotine.