Mesophyll protoplasts isolated from four sunflower species: H. annuus. H. giganteus, H. maximiliani, H. nuttallii were subjected to cultivation in liquid media. Various factors were analyzed and their optimization resulted in a high and reproducible percentage of viable and dividing cells in liquid culture. Donor plant pre-cultivation in the dark for two days, usage of 8-12 day-old leaves, selection of undifferentiated protoplasts, a cultivation density of 6 x 104 protoplasts ml" and cultivation in day/night cycle were found to be essential for a high and synchronous division rate of more than 70% dividing cells. Cell division was obtained for all four species at different rates reflecting their cultivation potential.