Both cholesterol and polyunsaturated fatty acid (PUFA) metabolism play an important role in retinal and brain development and function. Dietary intake of cholesterol is accompanied with higher risk of heart disease and was suggested to have a role in the pathogenesis of Alzheimer's disease, while dietary PUFAs were reported to act in an opposite way. The same phenomena could be seen in case of inflammation. These effects are mainly realized through gene expression changes. In the present study, the effects of dietary cholesterol and the combination of cholesterol and fish oil were analyzed on the modulation of fatty acid composition and gene expression in the brain and in the eye. At the transcription level, specific changes could be detected in both tissues among transcription factor genes coding for sterol regulatory element binding proteins, retinoid X receptors and peroxisome proliferator-activated receptors, and different fatty acid binding protein genes by using quantitative real-time PCR. In the eye, cholesterol diet attenuated the positive effects of fish oil on inflammatory gene expression as the combined diet resulted in increased RNAm level of phospholipase A-2, inducible nitric oxide synthase, TNF-alpha, COX-1, COX-2 and cytokine, ICAM-1. This induction was absent in the brain. Complex changes could be also recorded in the fatty acid composition of lipids extracted from eye and brain tissue due to the dietary intervention. One of the most interesting changes was the reduced level of docosahexaenoic acid by cholesterol in the eye. Our results on fatty acid composition and gene expression changes may open up new alleys in understanding the complex roles of cholesterol and PUFAs in normal and pathological visual and brain function.