In order to study the effect of vessel closure type and number of subculturing on the rate of proliferation and vitrification of carnation (Dianthus caryophyllus L.) cultivars (Mondeo Kgr and Prado Aquila Kgr), an experiment was conducted as factorial in completely randomized design with 5 replications. Lateral bud explants were cultured on MS medium supplemented with 2 mg/l BA and 0.1 mg/l NAA. Treatments were comprised of vessel closure type (foil, parafilm, selefon and paper), number of subculturing (culturing of explants and 2 stage of subculturing) and cultivar. Results showed that the highest amount of regenerated shoots was observed in second subculturing and foil closure for Prado Aquila Kgr (8.4) and in first subculturing and parafilm closure for Mondeo Kgr (8.6). Also in both cultivars the highest percentage of vitrification were observed in second subculturing and foil closure and the lowest of vitrification was observed in first culturing of lateral buds and paper closures. However, regenerated plants in selefon and paper closures had no good quality. But application of parafilm closure leads to increasing the rate of regeneration and reducing vitrification. Because of abnormal growth, vitrified plantlets have low percentage of surviving and they fail to acclimatized, so by considering the amount of multiplication and vitrification for obtaining the highest number of shoots, application of parafilm closure in first subculturing and foil closure in second subculturing of Mondeo Kgr and Prado Aquila Kgr is suggested, respectively.