[1,4-(14)C]Putrescine, administered to cultured roots of Hyoscyamus albus L., was converted to spermidine and spermine as well as to conjugated forms. A substantial fraction of its radioactivity (approx. 6%) was, however, incorporated into N-methylputrescine within 4 h, followed by an increase in radioactive tropane alkaloids (mostly hyoscyamine). Incubation of the roots with DL-[5-(14)C]ornithine and L-[2,3-(3)H]arginine separately resulted in rapid incorporation of each label into free putrescine and N-methylputrescine, followed by incorporation into hyoscyamine. During active alkaloid biosynthesis in H. albus roots, scarcely any radioactivity was recovered in δ-N-methylornithine. When the roots had been treated with 5 mM DL-α-difluoromethylornithine for 1 d prior to the administration of labeled ornithine, the total uptake of radioactivity into the root cells was not reduced, even though ornithine decarboxylase was specifically inactivated and the incorporation of the label to putrescine, N-methylputrescine, hyoscyamine and scopolamine markedly reduced. These and other results (Hashimoto et al., 1989, Planta 178, 123-130) are strong indications that tropane alkaloids in H. albus root cultures are synthesized from both ornithine and arginine by way of putrescine.