Root cultures of Plumbago rosea Linn. were established from young leaf explants on solid Gamborg's B5 (B5) medium supplemented with the combination of α-naphthalene acetic acid (NAA) and kinetin in the concentration ranges of 0.5-2.0 mg/l and 0.1-0.5 mg/l, respectively. The production of plumbagin, determined by TLC-densitometry was higher [0.016 ± 0.0030% dry weight (DW)] in cultured roots obtained from B5 medium supplemented with 1.0 mg/l NAA and 0.1 mg/l kinetin. Plant selection increased the plumbagin production to 0.129 ± 0.0139% DW, while variation of sucrose and nitrogen (as (NH₄)₂SO₄) concentration in B5 media slightly increase the plumbagin synthesis to 0.023 ± 0.0017 and 0.020 ± 0.0015% DW, respectively.