Palmitoleic acid is a minor monounsaturated fatty acid in the human diet and in blood plasma. Because macadamia oil is at least one potentially large source of palmitoleic acid, we tested its effect on plasma lipid levels against two other dietary fatty acids, oleic acid and palmitic acid. The dietary adjustments, through the use of supplements, provided comparisons of the three test fatty acids in which palmitoleic could be judged as behaving either like a saturated or a monounsaturated acid. Thirty-four hypercholesterolemic men ate the three test diets in random order in 3-week periods. Plasma total cholesterol and low density lipoprotein (LDL) cholesterol concentrations were similar with palmitic and palmitoleic acids and significantly higher than with oleic acid. High density lipoprotein (HDL) cholesterol was significantly lower with palmitoleic than with palmitic acid. The study confirms that, at least in hypercholesterolemic men, a modest increase in palmitic acid (+4% en) raises LDL cholesterol relative to oleic acid (+3% en), even when dietary cholesterol is low (< 165 mg/day). Palmitoleic acid (+4% en) behaves like a saturated and not a monounsaturated fatty acid in its effect on LDL cholesterol.