Long-chain polyunsaturated fatty acids (LCPUFAs), including docosahexaenoic acid ([DHA] 22:6 n-3), are important components of cell membranes. Low levels of DHA and other LCPUFAs in skeletal muscle membrane phospholipid are associated with insulin resistance and obesity in adults. These findings may be influenced by both dietary and genetic factors. This study aimed to investigate the interrelationships between the type of infant feeding, skeletal muscle phospholipid fatty acid (FA) composition, and glucoregulation in young children. Skeletal muscle biopsies and fasting blood samples were obtained from 56 normally nourished young children (35 males and 21 females) aged less than 2 years (mean +/- SE, 0.76 +/- 0.06) undergoing elective surgery. The dietary history was taken, and muscle phospholipid FA composition was analyzed. Subgroups of totally breast-fed and age-matched formula-fed infants were compared. Breast-fed infants (n = 13; age, 0.54 +/- 0.06 years) had a significantly higher percentage of DHA (3.63% +/- 0.22% v 1.84% +/- 0.11%, P < .0001) and total percentage of LCPUFAs (30.24 +/- 0.87% v 25.17% +/- 0.86, P < .0001) in muscle phospholipids compared with the formula-fed group (n = 12; age, 0.59 +/- 0.08 years). The totally breast-fed group had lower plasma glucose levels than the formula-fed group (4.7 +/- 0.2 v 5.4 +/- 0.2 mmol/L, P < .02). Consistent with these findings, further analysis of a group of 39 children who had either never or not recently been breast-fed showed significant inverse correlations between fasting plasma glucose and the percentage of both DHA (r = -.47, P < .003) and total LCPUFAs (r = -.38, P < .05). The results of this study show that (1) breast-feeding increases LCPUFA levels in skeletal muscle membrane and (2) early development of relatively higher levels of LCPUFAs in the phospholipid of skeletal muscle, influenced both by type of feeding and by genetic predisposition, is associated with lower fasting plasma glucose. Early changes in skeletal muscle membrane phospholipid FA saturation may play a role in the subsequent development of diseases associated with insulin resistance.