Ephedra is the most widely and largest widespread genus in the Gnetopsida, a subgroup of the gymnosperms. This research was done to find out how gamma radiation affected the in vitro growth of callus and suspension cultures. In addition, the effect of gamma rays (γ-rays) on the ephedrine and pseudoephedrine concentrations was evaluated through HPLC analysis. The cell suspension and callus cultures were formed on Murashige and Skoog's basal medium (MS) supplemented with 0.5 mg/l kinetin (Kn), and 3 mg/l of 2, 4-dichlorophenoxy acetic acid (2,4-D) and exposed to variable gamma radiation doses (0, 5, 10, 15, 20, 25 and 30 Gray). Highly significant differences in both fresh and dry callus weights were recorded due to the effect of gamma rays (γ-rays). The findings demonstrated that in the cell suspension and callus cultures, 15 Gray achieved the highest fresh and dry weights when compared to the control. Similarly, the highest concentrations of ephedrine and pseudoephedrine were found in suspension and callus cultures compared to control at 15 Gray.