Visceral and subcutaneous adipose tissue display important metabolic differences that underlie the association of visceral obesity with obesity-related cardiovascular and metabolic alterations. Recently, visfatin was identified as an adipokine, which is predominantly secreted from visceral adipose tissue both in humans and mice. In this study, we examined whether visfatin plasma concentrations (using enzyme immunosorbent assay) and mRNA expression (using RT-PCR) in visceral and subcutaneous fat correlates with anthropometric and metabolic parameters in 189 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Visfatin plasma concentration correlates positively with the visceral visfatin mRNA expression (r(2) = 0.17, P < 0.0001), BMI (r(2) = 0.062, P = 0.004), percent body fat (r(2) = 0.048, P = 0.01), and negatively with subcutaneous visfatin mRNA expression (r(2) = 0.18, P < 0.0001). However, in a subgroup of 73 individuals, in which visceral fat mass was calculated from computed tomography scans, there was no correlation between plasma visfatin concentrations and visceral fat mass. We found no significant correlation between visfatin plasma concentrations and parameters of insulin sensitivity, including fasting insulin, fasting plasma glucose concentrations, and the glucose infusion rate during the steady state of an euglycemic-hyperinsulinemic clamp independent of percent body fat. Visfatin gene expression was not different between visceral and subcutaneous adipose tissue in the entire study group nor in selected subgroups. We found a significant correlation between visceral visfatin gene expression and BMI (r(2) = 0.06, P = 0.001) and percent body fat (measured using dual-energy X-ray absorptiometry) (r(2) = 0.044, P = 0.004), whereas no significant association between BMI or percent body fat and subcutaneous visfatin mRNA expression existed (both P >0.5). In conclusion, visfatin plasma concentrations and visceral visfatin mRNA expression correlated with measures of obesity but not with visceral fat mass or waist-to-hip ratio. In addition, we did not find differences in visfatin mRNA expression between visceral and subcutaneous adipose tissue in humans.