Choline acetyltransferase (ChAT) activity was measured in a fraction of endothelial cells obtained by collagenase digestion from previously isolated rat brain cortex capillaries. ChAT specific activity in the dissociated cells (0.264 nmol X mg protein-1 X min-1) was nearly 7 times higher than the activity measured in 'capillary-forming' cells (0.038 nmol X mg protein-1 X min-1). It is not known whether this increase was caused by ChAT enrichment of the former cellular fraction, and/or by a change in the specific activity of the enzyme during the isolation procedure, but the finding would be in favor of the intrinsic localization of cerebrovascular ChAT in endothelial cells. This would support the hypothesis of a role for locally synthesized ACh in the vasodilating response to noxious stimuli.