The effect of various inhibitors of polyamine biosynthesis on free and bound polyamine accumulation and on rhizogenesis was followed in thin layers excised from the stem of Nicotiana tabacum L. cv. Samsun. Trichloroacetic acid-soluble (TCA-soluble) free, TCA-soluble bound, and TCA-insoluble bound putrescine and spermidine accumulated throughout the culture period in control explants, reaching a peak on days 15–18. All the inhibitors tested depressed the rhizogenic process, though to a different extent, while leaving most of the explants viable. DL-α-difluoromethylornithine plus DL-α-difluoromethylarginine (DFMO + DFMA), specific irreversible inhibitors of ornithine- and arginine decarboxylases, respectively, almost totally inhibited rhizogenesis and polyamine accumulation. DFMO alone allowed a certain concentration of putrescine and spermidine to accumulate and rhizogenesis to occur in 20% of the explants. Cyclohexylamine (CHA), a competitive inhibitor of spermidine synthesis, did not have a strong inhibitory effect on root formation but depleted free and bound spermidine almost totally. Methylglyoxal-bis(guanyl)hydrazone (MGBG), an inhibitor of spermidine synthesis, caused a strong reduction in rhizogenesis but an accumulation of polyamines at the end of the culture period. No noticeable reversion of the rooting inhibition was observed by supplying the drug together with the polyamine whose synthesis was inhibited. The uptake and accumulation of exogenously supplied polyamines throughout the culture period was followed. The involvement of the three classes of polyamines in rhizogenic response is discussed in relation to the effect of the inhibitors of their biosynthesis.