Background: Fat deposition in muscle has been found to be related to metabolic risk. Objective: This study compared soleus intramyocellular lipid (IMCL) concentrations after consumption of weight-maintaining, controlled diets differing in total fat and fat type. Design: This study consisted of 3 phases of 25 d each in a crossover, controlled feeding design. The low-fat (LF) diet provided 30.8% and 5.2% of energy from fat and polyunsaturated fat (PUFA), respectively. Two higher-fat diets were tested: the high-fat (HF) diet provided 37.9% and 5.8% of energy from fat and PUFA, respectively, and the high-PUFA (HPUFA) diet provided 36.3% and 9.7% of energy from fat and PUFA, respectively. Twenty-four men and women [age range: 19-65 y; body mass index (in kg/m 2): 20-35] whose LDL and glucose concentrations were between 130 and 180 mg/dL and <126 mg/dL, respectively, completed all study phases. Results: IMCL content was 1.88 times as high after the HF diet (P = 0.005) and 1.71 times as high after the HPUFA diet (P = 0.002) as after the LF diet. There was no significant correlation between percentage fat mass or waist circumference and IMCL content. With pooled data from all diets, there was no significant correlation between IMCL content and insulin or glucose concentration. There was no significant difference in IMCL content in subjects with or without the metabolic syndrome or in subjects with LDL particle pattern A or B. Conclusions: Our results suggest that IMCL content is not modulated by dietary fat type but by total fat intake and that reducing fat intake effectively lowers IMCL. However, the metabolic implications of having lower IMCL concentrations are not clear. © 2007 American Society for Nutrition.