We examined short-term and long-term repeatability (reliability) of the fatty acid (FA) composition of plasma phospholipids and cholesterol esters (CEs). For short-term reliability, fasting blood samples of 34 subjects were collected three times, 2 wk apart, and in 24 subjects duplicate samples were collected during each visit. For long-term reliability, two fasting samples were collected in 50 subjects approximate to 3 y apart. In both phospholipids and CEs, short-term and long-term reliability coefficients were > 0.65 for the major plasma FAs (16:0, 18:0, 18:2n-6, and 20:4n-6), with the exception of 18:1n-9, but were generally lower for FAs that compose < 1% of total FAs. Reliability tended to be better for CEs than for phospholipids. Method variability was small (< 5% of total variability for most FAs), indicating that biological and dietary variability contribute most to total variability. Plasma FA measurement warrants consideration as a biochemical marker of diet in epidemiologic studies.