OBJECTIVE To evaluate the associations between the usual intake of dietary fats and insulin concentrations. Insulin concentrations and insulin resistance have been positively related to risk for NIDDM, obesity, hypertension, dyslipidemia, and coronary heart disease, yet little is known of the environmental risk factors for relative hyperinsulinemia. Insulin resistance can be induced by high-fat feeding in laboratory animals; therefore, high-fat diets may increase risks for developing NIDDM. RESEARCH DESIGN AND METHODS Subjects included 544 nondiabetic women who participated in the second examination of the Kaiser Permanente Women Twins Study (1989-1990). Fasting and 2-h post 75-g glucose load insulin levels were determined. Dietary fat intake was assessed by a food frequency questionnaire. Generalized least-squares regression analyses for unpaired twin data were used to determine the relationship between dietary fat intake and insulin levels after adjustment for total calories, age, several behavioral variables, and in some models, percentage of body fat and waist-to-hip ratio. Associations of dietary fat intake with insulin levels were also evaluated within the subset of monozygotic twin pairs (n = 164 pairs) after removal of genetic influences by regression analysis of intrapair differences. RESULTS Among the 544 individual women, a 20 g/day increase in total dietary fat was associated with a higher fasting insulin level (9% [P < 0.001] before and 6% [P < 0.01] after adjustment for the obesity variables). Higher intakes of saturated fat, oleic acid, and linoleic acid were each positively related to higher fasting insulin values. The relation of dietary fat with fasting insulin was significantly attenuated among physically active women compared with those who were sedentary (P = 0.04), even after adjustment for obesity. Only saturated fat intake was significantly associated with 2-h postglucose load insulin level before (P = 0.04) but not after adjustment for obesity. Within identical twin pairs, total dietary fat was positively related to fasting insulin before (P = 0.03) but not after adjustment for obesity (P = 0.11). CONCLUSIONS High intake of total dietary fat is positively related to relative fasting hyperinsulinemia in nondiabetic women, particularly those who are sedentary. This effect appears to be partly mediated by the relation of dietary fat with obesity.