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Background: HDL cholesterol (HDL-C) can be divided into subfractions, which may have variable association with atherogenic process. There are mixed results about the association between HDL-C subfractions and risk factors for cardiovascular disease.Hypothesis: HDL-C subfractions have different associations with metabolic syndrome, low-grade inflammation and insulin resistance.Methods: 4,532 individuals between 35 and 74 years without previous cardiovascular disease not in use of fibrates were enrolled. HDL-C subfractions were separated by vertical ultracentrifugation (Vertical Auto Profile - in mg/dl) in HDL2-C and HDL3-C. HDL2-C/HDL3-C ratio, insulin resistance (HOMA-IR) and high-sensitivity C-reactive protein (CRP) were also included in the analysis.Results: The mean age was 51 ± 9 years, and 54.8% were women. In univariate analysis, HDL-C, HDL2-C, and HDL3-C were all inversely associated with each of the MetS defining factors, HOMA-IR values, and serum CRP. It was also observed a negative association between HDL2-C/HDL3-C ratio with the variables mentioned above even after adjusting for smoking, alcohol use, physical activity, and HDL-C levels (p <0.01).Conclusion: HDL-C and both subfractions are inversely associated with all the factors that define MetS, insulin resistance and low-grade inflammation. Additionally, the HDL2-C/HDL3-C subfractions ratio assessed by VAP is also significantly associated with the former factors even after further adjustment for total HDL-C and other confounding variables.