Brain laterality has been observed in animals and humans structurally, functionally, and behaviorally. MRI and CT scans have revealed pathological and normal brain asymmetry. A coarse assessment of rat or human brain fails to expose profound left/right differences, while a finer examination of its structure reveals an array of asymmetric features. This lateralization may be derived from evolutionary, genetic, developmental, epigenetic, and pathologic factors. However, brain structure and function is complex and macroscopic or microscopic asymmetries may be hard to discern from random fluctuations. This study concentrated on the hippocampus and we explored laterization employing a molecular high-throughput approach. Using proteomic analysis based on a combined approach of 2-D PAGE and MS, we examined differential protein expression in the hippocampi (left vs. right) of young adult male rats. Initial proteomic analysis demonstrated quantitative differences of approximately eighty proteins between the right (RH) and left hippocampus (LH). These were primarily energy-, cell metabolism-, stress-inducible chaperone proteins and cytoskeleton- proteins. Analysis revealed higher abundance of metabolic enzymes related to cellular energy metabolism, in the RH than the LH. In contrast, higher concentrations of proteins which are located mainly in astrocytes were shown in the LH than the RH. Immunoblotting of brain-specific proteins, on single animal hippocampal lysates confirmed the expression of Dynamin-1, DRP2, synapsin-1 and others, to be higher in the RH than LH lysates. These findings demonstrate major laterality in the expression of protein molecules between the two hippocampi providing a fertile field for mapping studies relating molecular, neuroimaging and functional data. Undoubtedly, asymmetries found at the animal level are hard to extrapolate to humans; however, studies in animal models will increase our understanding of the developing and adult brain and the healthy and diseased brain. © 2009 Wiley-Liss, Inc.