High‐density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL₂ and HDL₃, were isolated by two‐step discontinuous density‐gradient ultracentrifugation and the proteins were separated with two‐dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix‐assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A‐I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A‐II, apo A‐IV, apo C‐I, apo C‐II, apo C‐III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A‐IV (six isoforms). Furthermore, alpha‐1‐antitrypsin was identified in HDL for the first time. Additionally, salivary alpha‐amylase was identified as two isoforms in HDL₂, and apo L and a glycosylated apo A‐II were identified in HDL₃. Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A‐I and apo A‐II. Furthermore, the study reveals new proteins in HDL; alpha‐1‐antitrypsin and salivary alpha‐amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.