Peppermint (Mentha piperita L.) belongs to a member of the mint family (Lamiaceae) and is widely used in food, cosmetics and medicines. This study was carried to investigate the variation of essential oil content and its composition during callus subculture of M. piperita. For callus induction from the leaf explant of peppermint, the basal medium was supplemented with various concentrations of 2, 4-D. The best callus induction rate (93%) of M. piperita. was obtained in MS medium containing 2 mg/l 2, 4-D. The induced peppermint callus maintained on Lin-Staba medium were studied during a period of 20th subcultures for the stability of essential oil production. Growth rates of peppermint callus increased during prolonged subculture. However, there was a progressive decrease of essential oil content and unstability of monoterpene productions when callus cultures were serially subcultured.