To examine the relation between serum fatty acids and cancer, the authors conducted a nested case-control study of 108 middle-aged men who died of cancer and 215 men without cancer, who were enrolled in the Multiple Risk Factor Intervention Trial (MRFIT) between 1973 and 1976. Control subjects were matched to case subjects on age, smoking status, treatment assignment, date of randomization, and clinical center. After confirming the stability of the stored serum samples, the authors measured serum fatty acid levels by gas-liquid chromatography and analyzed their association with cancer. In stepwise logistic regression analyses that controlled for the MRFIT selection criteria variables and for alcohol consumption, no fatty acid was significantly associated with overall risk of cancer death (all p > 0.05). Serum levels of phospholipid dihomogammalinolenic acid (20:3), an essential fatty acid, were inversely associated with the risk of dying from lung cancer; a standard deviation increase was associated with a 32% decrease in risk (p = 0.05). Dietary cholesterol intake was associated with the risk of nonlung, non-digestive tract cancers; a standard deviation increase (331 mg/day) was associated with a 75% increase in risk (p = 0.02). The authors found no evidence to suggest that increased dietary intake or serum levels of polyunsaturated fatty acids were associated with an increased risk of fatal cancer among middle-aged men at high risk for coronary heart disease. The clinical significance of the inverse association between dihomogammalinolenic acid and lung cancer death is uncertain and requires confirmation.