Skeletal muscle lipoprotein lipase (LPL) overexpression in mice results in whole-body insulin resistance and increased intramuscular triglyceride stores, but decreased plasma triglyceride concentration and unchanged plasma free fatty acid (FFA) concentration. The effects of skeletal muscle LPL overexpression and fasting duration on FFA kinetics are unknown. Transgenic mice with muscle-specific LPL overexpression (MCKhLPL) and control mice (Con) were studied at rest during a 50-minute constant infusion of [9,10- 3H]palmitate to determine FFA kinetics after both 4 and 16 hours of fasting. FFA concentration was not different between groups after the 4-hour (Con, 0.80 +/- 0.06 mmol/L; MCKhLPL, 0.83 +/- 0.07 mmol/L) and 16-hour (Con, 0.83 +/- 0.04 mmol/L; MCKhLPL, 0.80 +/- 0.07 mmol/L) fast. FFA turnover (Ra) was not significantly different between MCKhLPL and Con groups after the 4-hour fast (Con Ra = 2.52 +/- 0.36 micromol/min; MCKhLPL Ra = 2.37 +/- 0.27 micromol/min). However, FFA turnover was significantly decreased after the 16-hour fast in MCKhLPL mice vs controls (Con Ra = 2.89 +/- 0.52 micromol/min; MCKhLPL Ra = 1.64 +/- 0.17 micromol/min; P < .05). The significantly lower FFA Ra in MCKhLPL vs control mice was due to a decrease in MCKhLPL FFA turnover from the 4- to 16-hour fast, whereas FFA turnover was unchanged in controls. The changes in FFA appearance after the 16-hour fast in MCKhLPL mice are most likely explained by increased reliance by skeletal muscle on plasma triglyceride as a fuel. These data suggest increased skeletal muscle LPL expression decreases dependence on plasma FFA during prolonged fasting in mice.