We have assessed the capacity of cultured protoplasts from two tissue sources of several commercially-grown broccoli cultivars to regenerate plants. A procedure that employs hypocotyl protoplasts and a culture medium with a high NAA:2,4-D auxin ratio was developed. The procedure permits highly efficient formation of colonies that regenerate shoots at frequencies of 8-17% with two of the four cultivars tested. The time required for the development of plants from protoplasts was 8-11 weeks. No mtDNA rearrangements were observed among any of 17 analysed regenerants. Double-stranded RNAs were detected in mitochondrial DNA (mtDNA) preparations of some, but not all, regenerants of one of the cultivars.