Abstract: Several factors, affecting the induction and proliferation of callus cultures of Thevetia peruviana Schum.were investigated in this study, including explants nature, the phyto-hormones (nature, concentration and combination) and the sucrose concentration. Nodal segments responded with higher frequency of callus induction and proliferation, as compared to inter-nodal segments and shoot apices, on MS medium supplemented with various concentration of 2,4-D and kinetin, individually. Among twenty five different growth regulators formulations supplemented to Murashige and Skoog’s medium, nodal segments responded with a maximum of 100% frequency of callus induction on a combination of 9.05 μM 2,4-D with 0.93 μM kinetin, followed by frequency of 88.3% on 6.97 μM Kinetin, supplemented alone. A very low frequency of callus induction, for all the three explants, was observed on media supplemented with different concentrations of 2,4-D alone. Maximum callus proliferation, resulting in fresh weight of 2.556 g, after one month of culture, was observed for callus initiated from nodal segments, on MS medium supplemented with 9.05 μM 2,4-D and 0.93 μM kinetin (the same medium giving highest frequency of callus induction). Of the three levels, sucrose at 3% was found as the most favorable level for callus initiation and proliferation. Callus could be successfully maintained in healthy and proliferative form for consecutive nine months on the MS medium supplemented with 9.05 μM 2,4-D and 0.93 μM kinetin.