We hypothesized that plasma non-esterified fatty acids (NEFA) are trafficked directly to intramyocellular long-chain acylcarnitines (imLCAC) rather than transiting intramyocellular triglycerides (imTG) on the way to resting muscle fatty acid oxidation. Overnight fasted adults (n = 61) received intravenous infusions of [U-(13)C]palmitate (0400-0830 h) and [U-(13)C]oleate (0800-1400 h) labelling plasma NEFA, imTG, imLCAC and im-non-esterified FA (imNEFA). Two muscle biopsies (0830 and 1400 h) were performed following 6 h, overlapping, sequential palmitate/oleate tracer infusions. Enrichment of plasma palmitate was approximately 15 times greater than enrichment of imTG, imNEFA-palmitate and im-palmitoyl-carnitine. Fatty acid enrichment in LCAC was correlated with imTG and imNEFA; there was a significant correlation between imTG concentrations and imLCAC concentrations in women (r = 0.51, P = 0.005), but not men (r = 0.30, P = 0.11). We estimated that approximately 11% of NEFA were stored in imTG. imTG NEFA storage was correlated only with NEFA concentrations (r = 0.52, P = 0.004) in women and with V(O(2),peak) (r = 0.45, P = 0.02) in men. At rest, plasma NEFA are trafficked largely to imTG before they enter LCAC oxidative pools; thus, imTG are an important, central pool that regulates the delivery of fatty acids to the intracellular environment. Factors relating to plasma NEFA storage into imTG differ in men and women.