Polyamines (PAs) and the cell wall have important roles in plant morphogenesis. An interaction between cell wall components (pectic substances) and polyamines is known and inhibition of polyamine biosynthesis induces inhibition of some morphogenic processes. This is the case of rhizogenesis in tobacco (Nicotiana tabacum L.) thin layers. This paper investigates whether inhibition of polyamine biosynthesis induces modifications in the structure, shape and size of the primary cell walls in tobacco thin layers cultured on a rhizogenic medium, and whether polyamines (putrescine and spermidine) administered in combination with their corresponding inhibitors DL-α-difluoromethylornithine +DL-α-difluoromethylarginine (DFMO+DFMA), cyclohexylamine (CHA), methylglyoxal-bis(guanylhydrazone) (MGBG) restore cell wall characteristics. In treatments with polyamine inhibitors, cell hypertrophy occurred in the lowermost layer of the explant in contact with the medium. Many unusual features were observed in the walls of the hypertrophic cells: an amorphous structure, loosening of the fibrillar component; detachment between contiguous cells, lysis of wall components and an uneven thickness often giving a wavy appearance (in transverse walls). The inhibitors reduced cell wall thickness, and caused a weak positive PATAg reaction for polysaccharides. Polyamines restored cell wall thickness and, in general, the other wall features. Our results show that polyamines are necessary for maintaining cell wall characteristics by strengthening the links between cell-wall components.