Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 105 pp ml−1. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.