The inhibition of human and mammalian red blood cell (RBC) cholinesterase (AChE) in whole blood in the presence of added pyridostigmine has been examined. After the addition of pyridostigmine to animal and human blood, red cells were separated from plasma at varying intervals and their enzyme activity measured. An apparent rate constant (ke) was derived for the reaction sequence in which carbamate is released from AChE inhibited by pyridostigmine. The constant is a complex of the rates of decarbamoylation and reinhibition of AChE in the blood sample. Rate constants were also determined for the spontaneous reactivation (ks) of carbamoylated AChE in the species studied. Values of Ks were greater than Ke in corresponding species but varied little between species. Pretreatment of animals with pyridostigmine is known to be an effective therapy against organophosphorus compounds, including soman. The ranking of ke values in mammalian blood was the same as that for the protection against soman in animals: monkey greater than guinea-pig greater than rabbit greater than rat (ke = 0.15, 0.07, 0.05, 0.02 h-1, respectively). Since ke for human blood (0.20 h-1) was greater than that of monkey, pyridostigmine pretreatment would be expected to be an effective prophylaxis for soman in humans.