Black cumin (Nigella sativa L.) seed oil was extracted with two different solvents, n-hexane (H) and a mixture of chloroform/methanol (CM) (2:1, by volume). Amount of total lipid (TL) was higher in the CM miscelle (39.2% of seed fresh weight) than in the H extract (37.9%). Chemical characteristics as well as fatty acid profile of the TL extracts were compared and the analysis revealed that the major fatty acid was linoleic acid C18:2n-6 (ca. 57% of total fatty acid methyl esters (FAME)) followed by oleic acid C18:1n-9. Palmitic acid C16:0 was the major saturated fatty acid and detected in appreciable level. Chromatography on a silica column with solvent of increasing polarity yielded 96.1-97.2% neutral lipids (NL) and ca. 3% of polar lipids. Gas liquid chromatography with flame ionization detector (GLC/FID) showed that the major fatty acid present in all lipid classes was C18:2n-6 followed by C18:1n-9 and C16:0 acids, respectively. Phospholipid (PL) classes were separated via normal-phase HPLC. Separation was achieved on a silica column by gradient elution from isooctane/2-propanol (6:8, by volume) to isooctane/2-propanol/water (6:8:0.6, by volume) lasting 35 min with UV detection at 205 nm. The major individual PL classes were found to be phosphatidylcholine (PC; ca. 46-48% of total PL) followed by phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), respectively. Phosphatidylglycerol (PG), lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC) were isolated in smaller quantities. The level of saturated fatty acids, namely palmitic C16:0 and stearic C18:0 acids, was considerably higher in PL classes than in the corresponding triacylglycerols. Characterization of PL profile from Nigella sativa L. seed oil as well as the development of new source of PL was the primary aim of this study.