In order to clarify effects of phytohormones on the viability and the cell wall regeneration of protoplasts isolated from Nicotiana tobacum L. var. BY4, protoplasts isolated from mesophyll tissue were cultured on the Murashige-Skoog liquid media supplemented with auxin (2,4-D, NAA, IAA) and/or cytokinin (kinetin, BAP, 2ip). Viability of protopplasts was higher in the culture medium containing auxin and cytokinin, especially in the combination of 2,4-D and BAP. The effectual cell wall regeneration of protolasts was observed when the protoplasts were cultured on the medium supplemented with auxin alone, especially with IAA. Cell wall regeneration started from 2-3 days after culture and was not detected at budding regions. When the protoplasts were cultured on the phytohormone-free medium, the viability of protoplasts dramatically decreased 4 days after culture.