The triglyceride content of skeletal muscle samples determined by lipid extraction correlates with the severity of insulin-resistant glucose metabolism in muscle. To determine whether this reflects increased triglyceride within muscle fibers and to test the-hypothesis that the lipid content in muscle fibers is increased in obesity, the present study was undertaken using quantitative histochemistry of Oil Red 0 staining of vastus lateralis muscle. A percutaneous muscle biopsy was performed in 9 lean subjects, 15 obese subjects without type 2 diabetes mellitus (DM), and 10 obese subjects with type 2 DM (body mass index [BMI], 23 .4 ± 1 .0, 33 .6 ± 0 .6, and 36.0 ± 1.1 kg. M⁻² for lean, obese, and DM, respectively). Eight obese and 7 DM subjects had a weight loss and reassessment of muscle lipid content. Transverse muscle cryosections were examined by light microscopy with quantitative image analysis (grayscale images obtained by analog to digital conversion) to determine a lipid accumulation index (LAI) based on the percentage of cross-sectional fiber area occupied by lipid droplets. Muscle fiber lipid content was greater in obese individuals with DM than in lean individuals (3 .62% ± 0.65% v 1 .42%: 0.28%, P < .05) but was not different in obese individuals without DM (2 .53% ± 0.41%). Weight loss reduced the LAI from 3 .43% ± 0.53% to 2.35% ± 0 .31%. In summary, lipid accumulation within muscle fibers is significantly increased in obesity and is reduced by weight loss. This provides important information regarding the accumulation and distribution of skeletal muscle triglyceride,in type 2 DM and obesity.