The purpose of the present study was to estimate the relative contributions of degradation and brain-to-blood elimination processes to the clearance of microinjected human amyloid-β peptide(1-40) (hAβ(1-40)) from mouse cerebral cortex, using a solid-phase extraction method together with a newly developed ultraperformance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) quantitation method for intact hAβ(1-40). The clearance rate constant of hAβ(1-40) in mouse cerebral cortex was determined to be 3.21 × 10(-2)/min under conditions where the saturable brain-to-blood elimination process across the blood-brain barrier (BBB) was expected to be saturated. Thus, this clearance rate constant should mainly reflect degradation. The [(125)I]hAβ(1-40) elimination rate across the BBB under nonsaturating conditions was determined to be 1.48 × 10(-2)/min. Inhibition studies suggested that processes sensitive to insulin and phosphoramidon, which inhibit neprilysin, insulin-degrading enzyme, and endothelin-converting enzyme, are involved not only in degradation, but also in elimination of hAβ(1-40). In conclusion, our results suggest a dominant contribution of degradation to cerebral hAβ(1-40) clearance, and also indicate that a sequential process of degradation and elimination of degradation products is involved in cerebral hAβ(1-40) clearance.Journal of Cerebral Blood Flow & Metabolism advance online publication, 21 August 2013; doi:10.1038/jcbfm.2013.125.