The activity of the acetylcholine (ACH) system per unit wt. was measured in samples of cerebral cortex from normal, unanesthetized adult mice, rats, guinea pigs, rabbits,cats, dogs, monkeys and beef and from human patients. The components detd. were: total ACH content, cholinesterase activity, and the rates of production of free and of bound ACH by slices incubated in media containing normal and high K concns. No obvious differences between different cortical areas in the same mammalian species were observed. The total ACH content of pieces of cerebral cortex decreased with time after excision. The rate of decrease was the same function of time for all species studied. The decrease depended on the condition of the tissue since excision and did not occur if the animal had been eserinized or if the excised pieces were kept under an increased oxygen tension. All the components of the ACH system measured decreased fairly regularly with ascending order on the phylogenetlc scale. This decrease was related to the avg. total brain wt. of each species and was the same function of the brain weight for all types of activity measured. A decrease in the avg. number of neurones per unit volume of cortex with increasing brain wt. ran parallel to the decrease in activities of the ACH system.