The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the δ-amino group or that decarboxylate δ-N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical δ-N-methylornithine.