We report a genetically encoded fluorescence lifetime sensor for protein kinase C (PKC) activity, named CKAR3, based on Förster resonance energy transfer. CKAR3 exhibits a 10-fold increased dynamic range compared to its parental sensors and enables imaging of PKC activity during animal behavior. Our results reveal robust PKC activity in a sparse neuronal subset in the motor cortex during locomotion, in part mediated by muscarinic acetylcholine receptors.