The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment "PLA") cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0. 5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0. 5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0. 5 μM NAA + 10 μM BA followed by a second culture on 0. 5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10. 0 μM NAA and 1. 0 μM gibberellic acid (GA3). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g-1 DW total FC) and lowest in organogenic calli (up to 50 μg g-1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage. © 2013 Springer Science+Business Media Dordrecht.