Protoplast fusion studies between various auxotrophic mutants of Nicotiana plumbaginifolia were performed to optimize conditions for PEG-mediated fusion and to identify factors influencing the plant protoplast fusion process. Numerous parameters in the isolation, culture, and fusion of protoplasts were tested, and established fusion protocols were compared. Fusion rates, calculated on the basis of colony growth on selection medium (genetic complementation), ranged from 10(-4) to 10(-2). Conditions that allow rapid and reproducible fusions at the highest rates were established. Particular emphasis was given to fusion of mesophyll-derived protoplasts, for which the ability to regenerate fertile plants from fusion products was shown to be particularly high. Preliminary experiments using electric-field mediated fusion suggest that electrofusion may offer significant advantages over the traditional chemical fusion.