A prospective study in healthy infants predefining both diet fatty acid and cholesterol, from birth to age 1 year, compared response of cholesterol fractions in three groups: random assignment to 1) monounsaturated-(Hi-Mono) (n = 20), or 2) polyunsaturated-(Hi-Poly) (n = 22) fatty acid-enriched diets, or 3) non-randomized selection to breast feeding (Human Milk) (n = 25). In each group, designated weaning foods and supplements maintained fatty acid and cholesterol intake similar to that of each group's defined formulas, with long-term compliance confirmed by plasma phospholipid fatty acid concentrations. By 12 months, total cholesterol was significantly lower in the Hi-Poly group compared to either of the other groups (P < 0.05). Low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol concentrations were significantly lower by 12 months in the Hi-Poly group, compared to the Hi-Mono groups. However, at the earlier 4-month interval, total cholesterol and LDL-cholesterol in both Hi-Mono and Hi-Poly groups were not different from each other, although each was significantly lower than the parallel Human Milk-group (P < 0.05). The Hi-Mono group increased gradually in total and LDL-cholesterol such that, after 12 months' feedings, all lipid fractions of this Hi-Mono group were no different from those of the Human Milk group. In independent group comparisons, there were no significant differences in HDL-cholesterol concentrations after 4 and 9 months on these diets. Independent of diet, HDL-cholesterol showed a falling trend as an overall time-effect across all groups (P < 0.001). These data suggest that prolonged feeding of a diet enriched in polyunsaturated acids in early infancy has a significant cholesterol-lowering effect compared to monounsaturates. These differences in total, LDL-, and HDL-cholesterol plasma concentrations between polyunsaturates and monounsaturates were not significantly evident until feedings had continued for a year.