IMPACT of different concentrations (0.25, 0.5 and 1mmol) of both alkaloids precursors,arginine (Arg) and ornithine (Orn) on production of hyoscyamine and its physiological attributes in 30 days old-callus of Egyptian henbane (Hyoscyamus muticus L.) was studied. Data of HPTLC revealed that in vivo wild leaves had high concentration of hyoscyamine but callus treated with 0.25mmol of Orn had more 3.2 and 1.4 time of total alkaloids and hyoscyamine than control, respectively. The same level of Orn induced the highest concentration of glutathione and reduced the H₂O₂ to the lowest rate. Although callus treated with 0.25mmol of Orn had only 6 visualized protein bands separated with SDS-PAGE, protein bands with low molecular weight 38, 33, 21, 15 and 9kDa were highly expressed. Callus cells exposed to low level of Orn had irregular amoeboid shape and lysiogenous intercellular spaces compared to regular ovate and schizogenous intercellular spaces in control. However, fresh and dry weight were increased under all levels of both precursors compared to control but water content % was maintained. Photosynthetic pigments and total carbohydrates were increased with increment of both Orn or Arg levels then decreased under high levels of each. Application of Orn at any levels decreased the content of free amino acids in contrary to Arg. Arg with high level induced the highest concentration of free phenolics and the activity of peroxidase. It was concluded that, addition of Orn with low concentration (0.25mmol) was efficient for hyoscyamine enhancement from Egyptian henbane callus than Arg.