It has been suggested that hippocampus-related cognitive processes are especially sensitive to ethanol. To provide an insight into the biochemical mechanisms underlying the hippocampus-related functional deficits associated with prenatal ethanol exposure, we investigated the effects of chronic ethanol exposure on the phospholipid profile in developing rat hippocampi. High-performance liquid chromatography/electrospray ionization–mass spectrometry analysis revealed that ethanol lowered the levels of total phosphatidylserine (PS) by 15–20% at all ages examined, primarily owing to the reduction in 1-stearoyl-2-docosahexaenoyl-PS (18:0,22:6n-3-PS) species. Ethanol exposure also led to a decrease in phosphatidylcholine (PC) and an increase in phosphatidylethanolamine (PE), but the total phospholipid content was not significantly changed. At the fatty acid level, ethanol exposure significantly decreased the 22:6n-3 content at postnatal days 0 and 21, with a slight increase in 22:5n-6, without changing the total fatty acid content significantly. In conclusion, ethanol depleted PS, especially 22:6-containing species, and PC from hippocampal membranes with concomitant increase in PE. Alteration of the phospholipid profile in the hippocampus resulting from exposure to ethanol during prenatal and developmental stages may have significant implications with respect to the cognitive dysfunction observed in fetal alcohol syndrome.