A highly-efficient protocol for the large-scale isolation of guard cell protoplasts from sugar beet (Beta vulgaris L.) has been developed. Optimization of conditions for culturing these protoplasts resulted in extensive cell division and colony formation, at frequencies exceeding 50%. Plants can subsequently be regenerated from these guard cell-derived colonies. This provides definitive confirmation that, in sugar beet leaf protoplast populations, only guard cells are the source of totipotent protoplasts. These findings are the outcome of a directed, non-empirical approach to overcoming plant cell recalcitrance which was initiated by exploiting computer-assisted microscopy to couple in vitro response to cell origin. The results reaffirm the conclusion that, in plants, extreme degrees of cytodifferentiation need not entail terminal specialization. The responsive nature of this system can be ascribed to the unique use of cultures essentially comprising a single in vivo cell type. A uniform model system has thus been created with potential for widespread application. Their distinct morphological (and mechanical) features make guard cells a valuable choice for studying various fundamental aspects, not only of stomatal physiology, but also of plant cell (de)differentiation, differential gene expression etc. Furthermore, an applied value for such a system can also be envisaged. Results indicate that these cells are highly amenable to genetic manipulation techniques. The importance of these observations to our understanding of plant cell function and behaviour is discussed.