Radioactive feeding experiments, in which DL-[pyrrolidine-2'-¹⁴C]-nicotine was added to 10 day old cultures, confirmed that the kinetic pattern of this N-demethylation was similar to that of non-radioactive nicotine, and that nornicotine was the major product and was produced intracellularly with a maximum percentage conversion of approximately 70%. The appearance of nornicotine paralleled the disappearance of the added nicotine, although small amounts of four other radioactive-metabolites were observed. One of these metabolites was tentatively identified by GC-MS as N-formyl-3-nornicotine. The properties of (-)-nornicotine produced from (-)-nicotine by 10 day old cell cultures were determined using both, polarimetry and chiral gas chromatography. The results obtained, which were convincingly consistent, showed that the nornicotine produced was exclusively one enantiomer. This provided strong evidence that the bioconversion of nicotine by tobacco cultures does not involve opening of the pyrrolidine ring. It is possible that the mechanism of nicotine bioconversion by cultured cells might differ from that proposed for the plant, in which a partially racemized mixture of (+)- and (-)-nornicotine has been reported. This could be the result of the opening and closing of the pyrrolidine ring during bioconversion. A radioactive enzyme assay procedure developed, using cell-free preparations of tobacco cell cultures, has shown for the first time that the enzyme(s) which catalyses this N-demethylation is present in tobacco. The enzyme(s), which has been fully characterised, has a Km of 7.4μM and a Vmax of 7.6 x 10^-2pKat and appears to be NADPH dependent. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity was present in the intermediate pellet whilst the maximum specific activity appeared to be associated with the microsomal fraction. Also, the addition of selected possible methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity, suggesting that the N-demethylation is unlikely to be a transmethylation but satisfies some of the primary criteria for cytochrome P-450 involvement. Studies involving Sephadex G-25 gel fractionation showed that molecules smaller than 5,000MW are not involved in this nicotine N-demethylation. Finally, the possible enzymatic mechanism involved in this N-demethylation is discussed.