Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by 13C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-13C] gamma-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6-10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. 13C NMR showed that 13C in gamma-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-13C]-arachidonic acid peaked in both brain and liver total lipids 48-96 h post-dosing. 13C enrichment in brain gamma-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48-96 h post-dosing was 1-2% of that in brain arachidonic acid. 13C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous gamma-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-13C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from gamma-linolenic acid is also incorporated into lipid products other than n-6 long-chain polyunsaturated fatty acids.