Kallikrein-6 and calpain-1 are amongst a small group of proteases that degrade ¿-synuclein. We have explored the possibility that reduction in the level or activity of these enzymes contributes to the accumulation of ¿-synuclein in Lewy body diseases. We measured calpain-1 activity by fluorogenic activity assay, kallikrein-6 level by sandwich ELISA, and levels of ¿-synuclein and ¿-synuclein phosphorylated at serine 129 (¿-synuclein-P129), in post-mortem brain tissue in pure dementia with Lewy bodies (DLB, n = 12), Alzheimer¿s disease (AD, n = 20) and age-matched controls (n = 19). Calpain-1 activity was significantly reduced in DLB within the cingulate and parahippocampal cortex, regions with highest ¿-synuclein and ¿-synuclein-P129 load, and correlated inversely with the levels of ¿-synuclein and ¿-synuclein-P129. Calpain-1 was unaltered in the thalamus and frontal cortex, regions with less ¿-synuclein pathology. Kallikrein-6 level was reduced in the cingulate cortex in the DLB cohort, and correlated inversely with ¿-synuclein and ¿-synuclein-P129. Kallikrein-6 was also reduced in DLB in the thalamus but not in relation to ¿-synuclein or ¿-synuclein-P129 load and was unaltered in the frontal and parahippocampal cortex. In SH-SY5Y cells overexpressing wild-type ¿-synuclein there was partial co-localisation of kallikrein-6 and calpain-1 with ¿-synuclein, and siRNA-mediated knock-down of kallikrein-6 and calpain-1 increased the amount of ¿-synuclein in cell lysates. Our results indicate that reductions in kallikrein-6 and calpain-1 may contribute to the accumulation of ¿-synuclein in DLB.