Purpose:Epidemiological studies suggest that dietary omega-3 polyunsaturated fatty acids (PUFAs) may protect against prevalence of dry eye. This work aimed to evaluate whether a dietary deficiency in omega-3 PUFAs may increase the severity of dry eye in a scopolamine-induced rat model. Methods:Three consecutive generations of Lewis rats were bred under diets deprived of omega-3 PUFAs. Dry eye was experimentally induced by continuous scopolamine delivery in female animals from the third deficient generation and in female Lewis rats fed for three generations with a balanced diet. After 14 days of treatment, the clinical signs of ocular dryness were evaluated in vivo using fluorescein staining. MHCII and the mucin Muc5AC were immunostained on ocular sphere cryosections. Lipids were extracted from the exorbital lacrimal glands and phospholipid fatty acids were analyzed by gas chromatography. Results:The ratio of fluorescein stained area to total area of the cornea was significantly increased in scopolamine-treated animals when compared to not implanted animals (from 2.4 ± 1.0% to 23.5 ± 13.6% for the balanced diet and from 7.8 ± 6.1% to 20.1 ± 6.2% for the deficient diet). Scopolamine treatment decreased Muc5AC immunostaining in the conjunctival epithelium (-34% for the balanced diet and -23% for the deficient diet) and tended to increase MHCII immunostaining in the conjunctival epithelium for both diets. In exorbital lacrimal gland phospholipids, arachidonic acid (AA) and the delta5-desaturase index (ratio of AA to dihomo gamma-linolenic acid) were significantly increased by scopolamine treatment for both diets. There was no significant diet-difference in scores of fluorescein staining, Muc5AC and MHCII immunostaining. The omega-3 PUFA deficiency induced a significant increase in AA in the exorbital lacrimal gland (+38% for the control animals and +15% for the scopolamine-treated animals).