Periwinkle (Catharanthus roseus) is one of the most important medical and ornamental plants in the world. In this investigation, periwinkle seeds, after sterilization were cultured on MS medium. Petiole segments of seedlings (4 day old) were subcultured to medium containing various concentrations of NAA accompanied with Kin and subcultured to regenerate the callus and root. Callus and roots were obtained from petioles in some of treatments. The extracts of callus and roots from different treatments were analyzed by spectrophotometer, TLC and HPLC with respect to the indole alkaloids producing capacity. Alkaloids were produced callus and roots from petiole of C. roseus in the presence of 0.1, 5, 10 and 20 mg/l Kin and NAA. MS with 0.1 mg/l NAA + 0.1 mg/l Kin had the highest vindoline, catharanthine, vincristine and root organogenesis capacity. But the level of these alkaloids and ajmalicine were very low compared to that in petiole of intact plant, and the level of serpentine was similar. New roots, callus roots, and callus from MS medium containing 0.1 mg/l NAA + 0.1 mg/l Kin were subcultured in hormone-free and 0.1 mg/l NAA + 0.1 mg/l Kin media and for organogenesis and growth. The most alkaloids amount was produced in new roots and callus roots. The indole alkaloid levels of new roots in new media were higher than in petioles of intact plants. In this study, 10-fold catharanthine, 125-fold serpentine, 0.5-fold vindoline and 0.34-fold ajmalicine were produced by new roots. The most interesting result was presentation of two important ant-cancer dimeric alkaloids, vinblastine and vincristine with amounts of 20-fold vinblastine and 6-fold vincristine to compare that in the petioles of intact plants.