Comprehensive studies were undertaken to determine optimal conditions for yellow-lupin (Lupinus luteus L.) protoplast culture. The adopted isolation procedure resulted in a high yield of protoplasts obtained from hypocotyls, cotyledons and young leaves. The usefulness of liquid and agarose-solidified media was evaluated in relation to promoting survivability and morphogenetic responses of protoplasts. The influence of different plant-growth regulators and anti-oxidative agents was also examined. Cultivar ‘Parys’ was the most promising material for manipulations. The solidification of medium enhanced the development of cultures initiated from hypocotyls and cotyledons, significantly increasing the division rate. In both liquid and solid media, mitoses were arrested after the initial division and daughter cells failed to divide. An important breakthrough in the development of cultures appeared in media supplemented with 0.1% activated charcoal. Bypassing the mitosis suppression led to the formation of small aggregates from hypocotyl protoplasts. This is the first report on protocolony formation in protoplast cultures of L. luteus. Notable progress achieved here indicates that, although this species is considered as recalcitrant to in vitro manipulations, it is possible to obtain responsive cultures with higher morphogenetic potential and thus provide new material for breeding programs of lupins.