Chelidonium majus L. is rich in many isoquinoline alkaloids which are responsible for the anti-inflammatory, antitumor and cytotoxic bioactivities known for this plant. The main alkaloids identified in C. majus L. include chelidonine, berberine, coptisine, sanguinarine and chelerythrine, which are present in different parts of the plant. In this work, alkaloids from C. majus aerial and terrestrial parts were extracted using a two step extraction procedure which consisted in a first step (SFE) using only supercritical carbon dioxide (scCO(2)) as solvent followed by a second step (ESE) using scCO(2) and a co-solvent mixture composed by an alcohol (ethanol or isopropanol) and diethylamine (alkaline conditions). The effect of operation temperature, pressure, solvent density and solvent pH on extraction yields, kinetic profiles and alkaloids' selectivity was evaluated. Results showed that SFE presents high selectivity for alkaloids (particularly for chelidonine) at solvent densities in the range 813-850 kg/m(3) and for both the aerial and terrestrial parts of the plant. The highest alkaloids extraction yield was observed at higher solvent density conditions using the scCO(2)/isopropanol/diethylamine mixture (ESE extraction step). Chelidonine was found to be highly soluble in scCO(2), Therefore, the fractioned high pressure extraction procedure proposed in this work can be successfully applied to separate enriched chelidonine fractions, while other alkaloids can be obtained using basified isopropanol as co-solvent. This procedure demonstrates that alkaloids fractionation during extraction is an important tool to be used before further purification/isolation steps. (C) 2016 Elsevier B.V. All rights reserved.