In the plant species that produce tropane alkaloids, two tropinone reductases (TRs) catalyze the stereospecific reductions of the 3-carbonyl group of tropinone. This reduction is a key branch point that determines the metabolite flow into the separate alkaloid groups, each with different stereospecific configurations. In this study, a specific antibody was prepared for each of the TRs by immunizing mice with recombinant TR protein and subsequent immuno-affinity purification of the antiserum. Immunoblot analyses revealed that accumulation of both TRs was highest in the lateral roots of Hyoscyamus niger throughout its development. In cultured roots, TR proteins were accumulated in a basal region but not in root apex. These patterns were similar to that of hyoscyamine 6 beta-hydroxylase (H6H), an enzyme that catalyzes a downstream step in the same biosynthetic pathway. However, an immunohistochemical analysis revealed that the two TRs and H6H were accumulated with different cell-specific patterns in the cultured root, suggesting transportation of the alkaloid intermediate(s) across the different cell layers.