Seeds of Pancratium maritimum collected from the natural populations along Bulgarian Black Sea coast were in vitro germinated in 2005 and cultures were maintained under equal conditions for three years (MS based medium with BAP and NAA; 23±2°C and 16/8 light/dark photoperiod). Directly regenerated bulblets with diameter of about 6–8 mm were cultured in parallel for two months on agar solidified and in liquid media containing 3 mg/L BAP and 0.1 mg/L NAA. Alkaloid determination by HPLC proved biosynthesis of lycorine which antiviral and antitumor properties are well known. Lycorine accumulation was up to 2.90 and 1.53 mg/g DW in the cultures grown on agar and in liquid medium, respectively, which was highly promising as first trials to produce this alkaloid in vitro. Additionally, bulblets were regenerated from callus cultures and comparison was done concerning the morphology of directly and indirectly obtained plantlets. Bulblet growth varied in accordance with the sucrose quantity in the medium as well.