Objective of the study is to estimate the effect of growth regulators and different ecotypes of Egyptian Catharanthus roseus such as (Ismailia, Fayoum, Alexandria, Sharkia and Cairo) on callus induction and alkaloids production. Different growth regulators combinations were applied. Murashige and Skoog (MS) medium supplemented with four different growth regulators, i.e. M1 (2, 4-Dichlorophenoxy acetic acid (2, 4-D) 1.0 μM and Benzyl adenine (BA) 0.5 μM), M2 (2, 4-D 0.5 μM and BA 1.0 μM), M3 (BA 1.0 μM and 1-Naphthaleneacetic acid (NAA) 0.5 μM) and M4 (BA 0.5 μM and NAA 1.0 μM). M1 medium gave the highest value for callus induction frequencies (75%) and callus fresh weight (1.87 g) for produced callus from leaf  while the  stem  produced callus (0.7 g) was the higher too than  other mediums. The total alkaloids content (TAC) was estimated using spectrophotometer with Bromocresol green (BCG). The results showed that TAC outperformed in produced callus from stem explant than leaf produced callus, leaf and stem of original plant. The important result showed that the Fayoum ecotype was the highest alkaloid content (2.22 mg/g) for produced callus from stem explant than all studied ecotypes.