Several factors were tested for their influence on the yield and viability of mesophyll protoplasts isolated from P. cerasus L. cv. Montmorency. As optimal conditions were determinated the development of good plants on medium “e” for 10 days. Suitable age of leaves for protoplasts isolations is 10 days cultures. The best viability (95%) of mesophyll protoplasts were obtained using enzyme solution I—Cellulose Onozuca RS—1%; Rhozyme HP—150–1%; Pectolyase Y—23–0.1%. We observed increasing of protoplasts yield and viability after auxin pre-treatment of the leaves. Cultural media based on Murashige and Skoog's (MS) salts supplemented with NAA 0.05 mg/l, BAP -0.5 mg/1 and Z—1 mg/l promoted cell wall regeneration (7th day), the first mitotic division (10–14 days), cell colonies (35 days) and formation the microcalli (65–70 days).