Study objective: To determine the effects of fish oil supplementation on plasma cholesterol in middle-aged men with isolated hypercholesterolemia. Design: Randomized double-blind placebo-controlled (safflower oil) two-period crossover trial with 12-week treatment periods. Setting: Outpatient general medicine clinic at a university-affiliated Veterans Affairs hospital. Patients: Thirty-eight men with plasma cholesterol between 5.68 and 7.76 mmol/L (220 to 300 mg/dL), triglyceride levels less than 3.39 mmol/L (300 mg/dL), and free of coexisting diseases. Interventions: Fish oil and placebo (safflower oil) supplementation. After basal measurements and a 4-week lead-in period, twenty 1-g capsules of either fish oil or placebo oil were provided for 12 weeks (period 1). After a 4-week washout phase participants then received the other oil for an additional 12 weeks (period 2). Measurements and main results: Blood was drawn at the beginning and end of each study period and analyzed for levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoprotein A1, and apolipoprotein B. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Total and LDL cholesterol increased from the before treatment values by 4.8% and 9.1%, respectively, after ingestion of fish oil. Compared with placebo, LDL cholesterol was significantly higher (4.5 compared with 4.1 mmol/L, P = 0.01) and triglycerides lower (1.3 compared with 1.8 mmol/L, P = 0.01) after fish oil. Total and HDL cholesterol and apolipoprotein A1 and B levels did not differ. Conclusions: Fish oil supplements do not lower plasma cholesterol levels in middle-aged men with hypercholesterolemia without elevated triglycerides. They should not be recommended as a method to lower plasma cholesterol in these patients.