Aim/hypothesis: The objective of this study was to describe the relation of serum fatty acids and desaturase activity (DA) to overweight, insulin sensitivity and cardiovascular disease (CVD) risk factors in adolescents. Methods: The relations of % serum phospholipid (PL) and cholesteryl ester (CE) fatty acids and estimated DA with CVD risk factors were examined in 264 adolescents (average age 15 years). Fatty acids were determined by gas liquid chromotography. Surrogate measures of DA were expressed as ratios of serum fatty acids: D9 DA ¼ 16:0/16:1; D6 DA ¼ 20:3,n6/18:2,n6 (PL) or 18:3,n6/18:2,n6 (CE); and D5 DA ¼ 20:4,n6/20:3,n6. Spearman partial correlations of fatty acids (%) and DA ratios with CVD risk factors were reported, adjusting for age, sex, race, Tanner stage, energy intake and physical activity. Results: Overweight adolescents compared to normal weight had more adverse levels of CVD risk factors, composition of PL and CE fatty acids in serum, and D6 DA and D5 DA ratios. Linoleic acid was inversely related to body mass index (BMI), waist circumference and triglycerides (Pp0.01). Dihomo-g-linolenic acid was positively related to BMI, waist, insulin, and triglycerides, and inversely related to high-density lipoprotein-cholesterol levels (Pp0.01). D6 DA was adversely associated with most of the risk factors (Pp0.01), whereas triglycerides and fasting insulin were beneficially related to D5 DA (Pp0.01). Conclusion: These findings support those observed in adults, that factors, such as type of dietary fat, physical activity, and obesity, may influence fatty acid metabolism and are important in the development of adverse CVD risk factors as early as adolescence.