Shoot regeneration was achieved from stored mature cotyledons of sweet cherry (Prunus avium L.) in vitro. The influences of different cytokinins (thidiazuron (TDZ) and benzylaminopurine (BA)) and their levels, the dark incubation for the first 10 days of the culture and TDZ pretreatments on shoot regeneration were determined. All varieties regenerated in the presence of TDZ if cotyledons were maintained in darkness for the first 10 days of the culture; however, only three varieties regenerated with low frequencies in the absence of dark incubation. Dark incubation at the early stage of culture was critical for obtaining higher regeneration efficiencies from stored cherry cotyledons. TDZ was more effective than BA in inducing shoot regeneration. The highest regeneration efficiencies were obtained with intermediate concentrations of TDZ (3.6 and 7.2 mu M) in combination with dark incubation and the best regeneration frequencies for 'Vista', 'Sunburst', 'Tehranivee', Nouge' and 'Heidelfingen' cotyledons were 70.0%, 53.3%, 23.3%, 30% and 26.6%, respectively. (c) 2007 Elsevier B.V. All rights reserved.