5-Pregnen-3beta-ol-20-one (pregnenolone), 4-pregnene-3,20-dione (progesterone), 5-pregnene-3beta,21-diol-20-one (21-hydroxypregnenolone), 4-pregnen-21 -ol-3,20-dione (cortexone), 5beta-pregnane-3,20-dione, 5alpha-pregnane-3,20-dione, 5beta-pregnan-3alpha-ol-20-one, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta, 21-triol-20-one 3-acetate, 23-nor-5-cholenic acid-3beta,20xi-diol, and 23-nor-3,20(22) E-choladienic acid-3beta-ol were administered to photomixotrophic shoot cultures of Digitalis lanata Ehrh. capable of synthesizing cardenolides, as well as to cardenolide-free tissue cultures, such as auxotrophic, dark-grown shoot cultures and cell suspension cultures of the same plant species. None of the pregnane precursors was qualified to restore cardenolide biosynthesis in the cardenolide-free tissues. The cardenolide content of light-grown shoot cultures, on the other hand, increased by 161%, 240%, 30%, 430% and 80% when 100 mg l(-1) of 21-hydroxypregnenolone, 5beta-pregnane-3,20-dione, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta,21-triol-20-one, 23-nor-5,20 (22) E-choladienic acid-3beta-ol, respectively, were administered. Pregnenolone, progesterone, cortexone, 5alpha-pregnanes, 5beta -pregnan-21-ols, and 23-nor-5-cholenic acid-3beta,20xi-diol, on the other hand, had no visible effect. Two different types of cardenolides (termed fucose-type cardenolides and digitoxose-type cardenolides) were identified which may be formed via different biosynthetic routes. The "norcholanic acid pathway" seems to be operative in D. lanata shoot cultures only in the formation of fucose-type cardenolides.