Technical Abstract: The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of B-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of the Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and the Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene was accumulated at comparable levels and no major difference in the carotenogenic gene expression was observed between the wild type and Or calli. These results suggested that the Or-induced B-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.