Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.