A drastic increase in rosmarinic acid (RA) accumulation is induced in cultured cells of Lithospermum erythrorhizon after their exposure to yeast extract or methyl jasmonate. Addition of the elicitors to the cell cultures only slightly enhanced the activity of rosmarinic acid synthase, which catalyzes formation of 4-coumaroyl-4′-hydroxyphenyllactic acid (CHPL), the first reaction specific to RA biosynthesis. In contrast, the two-step cytochrome P450-catalyzed hydroxylations of CHPL to form RA were dramatically up-regulated by the elicitor treatments, indicating that these hydroxylation activities are likely to play a key regulatory role in elicitation of RA biosynthesis.