High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.