Intramuscular triglyceride (IMTG) deposition in skeletal muscle is associated with obesity and type 2 diabetes (T2DM), and thought to be related to insulin resistance (IR). Curiously, despite enhanced skeletal muscle insulin sensitivity, highly-trained athletes and calorie-restricted (CR) monkeys also have increased IMTG. Sterol regulatory element binding proteins (SREBP) are transcription factors that regulate the biosynthesis of cholesterol and fatty acids. SREBP-1 is increased by insulin in skeletal muscle in vitro, and in skeletal muscle of IR subjects, but SREBP-1 expression has not been examined in exercise training or CR. We examined the relationship between IMTG and SREBP-1 expression in animal models of exercise and CR. Methods/Results: Gastrocnemius and soleus muscle biopsies were obtained from 38 Sprague Dawley rats (18 control and 20 exercise-trained). Triglyceride content was higher in the gastrocnemius and soleus muscles of the trained rats. SREBP-1c mRNA, SREBP-1 precursor and mature proteins, and fatty acid synthase (FAS) protein were increased with exercise training. Monkeys (Macacca mulatta) were CR for a mean of 10.4 years, preventing weight gain and IR. Vastus lateralis muscle was obtained from 12 monkeys (6 CR and 6 controls). SREBP-1 precursor and mature proteins, and FAS protein were higher in the CR monkeys. In addition, phosphorylation of ERK1/ERK2 was increased in skeletal muscle of CR animals. Summary: SREBP-1 protein and SREBP-1c mRNA are increased in interventions that increase IMTG, despite enhanced insulin sensitivity. CR and exercise-induced augmentation of SREBP-1 expression may be responsible for the increased IMTG seen in skeletal muscle of highly conditioned athletes.