Abstract.Guard cells are uniquely differentiated to transduce signals into the metabolic and ion transport processes that result in turgor‐driven stomatal movements. We tested the hypothesis that these highly specialized cells are terminally differentiated. Guard cell protoplasts were isolated from abaxial epidermal tissue of leaves ofNicotiana glauca(Graham) and cultured in a medium designed for culturing mesophyll protoplasts ofNicotiana tabacum.Protoplasts were incubated at densities of 2–5 × 1011cells m−3in eight‐well microchamber slides under 50μmol m−2s−1of photons of continuous fluorescent light at 25°C. When the medium was modified by the addition of 100mol m−3of sucrose and by buffering with 10mol m−3of MES buffer at pH 6.1, cell division began within 96h of the time the culture was initiated. After 9d of culture, 80% of surviving cells had synthesized new cell walls, had dedifferentiated, and were dividing to form small colonies. Callus tissue was visible after 4–5 weeks. We conclude that guard cells ofNicotiana glaucaare not terminally differentiated, and that guard cell protoplasts of this species have the capacity to grow, synthesize c