Mesophyll protoplasts of Nicotiana tabacum cv. Xanthi were subjected to centrifugation through a Percoll gradient. This resulted in the removal of the central vacuole from each protoplast, and improved mechanical and osmotic stability. EM studies showed that the remaining cell contents, including small vacuoles, were of normal morphology. Fixation of protoplasts at various times during subsequent culture showed that the central vacuole was restored after .apprx. 12 h. Cell-wall formation was well advanced after 24 h of culture. Results are discussed in terms of the potential use of evacuolate protoplasts and the mechanisms of vacuole formation.