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.