Silymarin production was examined in cell suspension cultures derived from hypocotyl of Silybum marianum (L.) Gaertn. MS medium supplemented with different concentrations of sucrose, glucose, KH₂PO₄, KH₂PO₄ + K₂SO₄, NH₄NO₃, CaCl₂, L-Phenylalanine, L-Tyrosine and yeast extract was used. Tyrosine 10 mg/l,  0.5 KH₂PO₄ + K₂SO₄ con., 0.75 NH₄NO₃ con., 0.75 KH₂PO₄ + K₂SO₄ con., glucose 30 g/l, 0.5 CaCl₂.2H₂O, tyrosine 20 mg/l and yeast extract 40 mg/l (3 days incubation period) showed the highest production of silymarin in callus cells to reach 79%, 57.5%, 46%, 24.2%, 21.2%, 19.7%, 9.7% and 8.6% over the control respectively. The same trend was obtained by HPLC (qualitative assay).