Four barbiturates, barbituric acid, butethal, phenobarbital, and 2-thiobarbituric acid, of fourteen tested were found to induce accumulation of benzophenanthridine alkaloids in cell suspension cultures of the California poppy Eschscholzia california. When the plant cell suspension cultures were treated with 1 mM barbiturate, alkaloids accumulated to 100 mg/l within four days. This is a level comparable to that achieved with 300 microM concentration of the established secondary metabolite inducer methyl jasmonate. In contrast to methyl jasmonate, barbituric acid, and 2-thiobarbituric acid, butethal and phenobarbital treatment resulted in a different alkaloid profile, suggesting that only select cytochrome P-450 genes were activated by these latter two barbiturates. RNA gel blot analysis of barbiturate induced cell cultures confirmed that transcripts of at least two benzophenanthridine alkaloid biosynthetic genes cyp80b1 (encoding the cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase) and bbe1 (encoding the covalently flavinylated berberine bridge enzyme) increased up to 5- to 7-fold over control values.