Protoplasts obtained from cell suspensions of an anthocyanin synthesizing strain ofDaucus carota cv. “Rote Riesen”, cultured on a modified Murashige and Skoog medium (MS) have been induced to regenerate cell walls, and divide repeatedly to form masses of callus. Techniques have been substantially refined, and optimal conditions for the isolation of protoplasts have been established. An optimal protoplast yield of 80–90% was obtained by treating the cells with 1.5% cellulase (pH 5.0) for 4.5 hours in a gently shaking water-bath maintained at 33 °C. The protoplasts when plated in the agar-solidified medium regenerated cell walls within 2 days, and first division was observed within 3 days. In addition, they showed irregular elongation, budding and the formation of sub-protoplasts; another phenomenon was the formation of a chain of buds, which either separated from each other or formed a “coenocytic”, tube-like structure. On the agar medium which contained 0.425 M sorbitol, growth slowed down or stopped after 3–4 weeks. In order to obtain further growth, it was necessary to transfer small pieces of agar containing protoplasts on to the top of a fresh medium containing lower amounts (0.2 M) of sorbitol. The protoplasts continued to divide to form cell colonies, and finally masses of callus after 4–5 weeks. In a liquid medium (B5) the protoplasts reacted similarly, they regenerated walls within 2 days and after 2 weeks anthocyanin containing callus was formed. In both media embryo-formation occurred after 6 weeks of culture. By combining the polyethylene glycol and the high pH fusion techniques intergeneric fusion was achieved between carrot protoplasts and mesophyll protoplasts from haploidNicotiana tabacum cv. “Badischer Burley”. The fused products could be readily identified and isolated by using the difference in colour as the visual markers. Conditions for the culture of these fused protoplasts are being worked out.