Apolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer's disease (AD). ApoE peptides are biologically active and may be produced in the brain. It is unclear if apoE proteolysis is dependent on isoform or AD status and this was addressed here. Hippocampus, frontal cortex, occipital lobe and cerebellum samples were homogenized into fractions that were soluble in Tris-buffered saline (TBS), Triton X-100 or guanidine hydrochloride and analysed for apoE fragmentation by Western blotting. Approximately 20% of apoE3 was detected as fragments and this was predominantly as a 25. kDa peptide in TBS-soluble fractions. The concentration of TBS-soluble apoE fragments was two- to three-fold higher in apoE3 compared to apoE4 subjects. This difference was observed in all areas of the brain examined and was not related to AD status. Cathepsin-D treatment generated apoE fragments that were very similar to those detected in brain, however, no apoE isoform-specific differences in susceptibility to cathepsin-D proteolysis were detected. This indicates that proteolytic processing of apoE to form soluble fragments in the human brain is dependent on apoE isoform but not AD status. © 2009 Elsevier Inc.