Protoplasts prepared from suspension-cultured Vinca rosea cells were grown in a liquid medium, and the effects of osmolarity and growth regulators on cell division and on the composition of regenerated cell walls were investigated. The concentration of mannitol optimal for cell division was 0.3-0.4 M. The presence of 2,4-D was essential for cell division, and 6-benzylaminopurine (BAP)enhanced cell division at concentrations of 0.03-0.1 ppm. However, the composition of regenerated cell walls was abnormal under suspension culture; the predominant sugar was glucose, indicating that the regenerated cell walls consisted mostly of glucans, and that the other cell-wall components were released into the medium. Mannitol, 2,4-D, and BAP at various concentrations did not significantly affect the sugar composition of the regenerated cell walls. Compared with liquid culture, cell division was stimulated when protoplasts were cultured on agar, and their regenerated cell walls had a composition similar to that of the original culture. The importance of the physical environment for the deposition of polysaccharide components in cell walls and the interrelationship between cell division and the regeneration of cell walls by protoplasts are discussed.