Anthocyanins are secondary metabolites that contribute to colors of flowers, fruits and leaves. Asiatic hybrid lily (Lilium spp.) accumulates cyanidin anthocyanins in flower tepals, tepal spots and leaves of juvenile shoots. To clarify their mechanisms of regulation of anthocyanin pigmentation, two full-length cDNAs of R2R3-MYB (LhMYB6 and LhMYB12) were isolated from the anthocyanin-accumulating tepals of cultivar 'Montreux'. Analysis of the deduced amino acid sequences indicated they have homology with petunia AN2, homologous sequences of which had not been isolated in species of monocots. Yeast two-hybrid analysis showed that LhMYB6 and LhMYB12 interacted with the Lilium hybrid basic helix-loop-helix 2 (LhbHLH2) protein. Transient expression analysis indicated that co-expression of LhMYB6 and LhbHLH2 or LhMYB12 and LhbHLH2, introduced by a microprojectile, activated the transcription of anthocyanin biosynthesis genes in lily bulbscales. Spatial and temporal transcription of LhMYB6 and LhMYB12 was analyzed. The expression of LhMYB12 corresponded well with anthocyanin pigmentation in tepals, filaments and styles, and that of LhMYB6 correlated with anthocyanin spots in tepals and light-induced pigmentation in leaves. These results indicate that LhMYB6 and LhMYB12 positively regulate anthocyanin biosynthesis and determine organ- and tissue-specific accumulation of anthocyanin.