The objective of this study was to investigate whether short-term zinc deficiency in the early neonatal period would exacerbate the effects of essential fatty acid (EFA) deficiency on liver and brain long-chain polyunsaturated fatty acid (LCPUFA) composition, as well as on behavioral development in artificially reared rat pups. Using a 2 × 2 factorial design, male Long-Evans rat pups were reared artificially from postnatal d 5 to 16; pups were fed through gastrostomy tubes with rat formula deficient in zinc and/or EFA. As expected, EFA deficiency significantly reduced levels of arachidonic acid [AA, 20:4(n-6)] and docosahexanoic acid [DHA, 22:6(n-3)] in liver phosphatidylcholine (PC) and brain phosphaditylethanolamine (PE), and increased 22:5(n-6) levels in liver and brain PC and PE. There were significant interactions between zinc and EFA in liver such that zinc deficiency reduced AA and DHA in the EFA-adequate groups, but significantly increased AA in the EFA-deficient groups. Contrary to the hypothesis, short-term zinc deficiency did not exacerbate the effects of EFA deficiency in liver phospholipids. In brain PE, a significant interaction between EFA and zinc was observed such that zinc deficiency increased 22:5(n-6) concentrations in EFA-adequate but not in EFA-deficient groups. Regardless of their EFA status, zinc-deficient rats were growth retarded and demonstrated deficits in locomotor skills. Possible effects of long-term zinc and EFA deficiency on brain function should be investigated in future studies.