In patients with familial combined hyperlipidemia (FCHL) and type 2 diabetes (DM2) organ-specific differences in insulin resistance may exist. In FCHL and DM2 in vivo insulin mediated muscle glucose uptake and inhibition of lipolysis were studied by euglycemic hyperinsulinemic clamp. Insulin mediated glucose uptake was impaired to the same extent in both FCHL and DM2. Only FCHL subjects showed no reduction in plasma glycerol concentrations during insulin infusion and incomplete suppression of plasma free fatty acid (FFA) concentrations combined. This finding indicated that insulin-induced suppression of lipolysis, or glycerol/FFA utilization, or both, were impaired in FCHL, in contrast to DM2 or control subjects. To analyze these possibilities in more detail, control, FCHL, and DM2 adipocytes were studied in vitro. In contrast to adipocytes from DM2 or control subjects, no reduction in medium FFA concentration was detected with FCHL adipocytes after incubation with insulin. This finding indicated impaired intracellular FFA utilization, most likely impaired FFA re-esterification. Genetic linkage analysis in 18 Dutch families with FCHL revealed no evidence for involvement of LIPE, the hormone sensitive lipase gene, indicating that genetic variation in adipocyte lipolysis by LIPE is not the key defect in FCHL. In conclusion, FCHL as well as DM2 subjects exhibited in vivo insulin resistance to glucose disposal, which occurs mainly in muscle. FCHL subjects showed insulin resistant adipose tissue lipid metabolism, in contrast to DM2 and controls. The different pattern of organ-specific insulin resistance in FCHL versus DM2 advances our understanding of differences and similarities in phenotypes between these disorders.