Methylphosphonic difluoride (difluoro) and its hydrolysis products, methylphosphonofluoridate (MF) and fluoride, were examined for cholinesterase-inhibiting ability in rats and guinea pigs by both inhalation and intraperitoneal exposure routes. In vivo inhibition was compared to in vitro inhibition. In the whole animal, MF was the active chemical, but in vitro under special conditions, difluoro was more potent than MF and fluoride. Rat and guinea pig blood cholinesterase were equally sensitive to inhibition by MF, but only the guinea pig displayed cholinergic signs leading to death from MF toxicity. Data imply that MF is responsible for the cholinesterase inhibition resulting from exposure to DF vapor. MF may be the first example of a moderately strong acid shown to inhibit cholinesterase and cause death from cholinergic effects.