Human amniotic fluid and a mixture of eight fatty acids (FAT-M) identified in this maternal fluid (C12:0, lauric acid, 0.9 μ g%; C14:0, myristic acid, 6.9 μ g%; C16:0, palmitic acid, 35.3 μ g%; C16:1, palmitoleic acid, 16.4 μ g%; C18:0, stearic acid, 8.5 μ g%; C18:1 cis, oleic acid, 18.4 μ g%; C18:1 trans, elaidic acid, 3.5 μ g%; C18:2, linoleic acid, 10.1 μ g%) produce anxiolytic-like effects that are comparable to diazepam in Wistar rats, suggesting the involvement of γ -aminobutyric acid-A (GABA A) receptors, a possibility not yet explored. Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABA A receptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg), GABA A benzodiazepine antagonist flumazenil (5 mg/kg), and noncompetitive GABA A chloride channel antagonist picrotoxin (1 mg/kg). The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABA A antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABA A receptor chloride channels.