A high-frequency in vitro regeneration of Digitalis davisiana Heywood (Alanya foxglove) and cardiotonic glycoside production from both in vitro produced materials (regenerated plantlets or germinated seedlings) and leaves of natural populations were obtained. Cardiac glycosides regulate heart rhythm and are effective in cancer chemotherapy, in particular for prostate and breast cancer treatments. Testing six different types of culture media revealed that Linsmaier and Skoog (LS) was the most effective for shoot production. Shoot regeneration efficiency was higher when flamingo-bill or hypocotyl explants were cultured on LS medium containing 0.5 mg/l thidiazuron (TDZ) and 0.25 mg/l indoleacetic acid (IAA). Rooting of all shoots that regenerated was readily achieved, even in the absence of plant growth regulators (PGRs). Production of cardenolides (lanatoside C and digoxin) in the materials grown in vitro seemed to correlate with several parameters, such as nutritional and hormonal compositions of the culture medium as well as the duration of culture on the initial regeneration and/or final growth medium. Higher amounts of digoxin accumulation were obtained when shoots were regenerated on LS or Gamborg's B5 medium containing 0.5 mg/l TDZ and 0.25 mg/l IAA, producing 12.59 and 11.93 mg/kg dry weight (dw) digoxin, respectively. For natural populations, seasonal variations seemed to affect the production of digoxin in the leaves. The highest amount of digoxin (246.58 mg/kg dw) was in leaf samples collected in July, which coincides with the flowering stage of the plant in the region of collection.