The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L.mmol-1.cm-1) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (-)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (-)-psi-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (-)-cocaine was a poor substrate for debenzoylation, it was a fairly good competitive inhibitor (Ki approximately 10 microM) of the hydrolysis of other substrates. The cocaine metabolites (-)-norcocaine, (-)-benzoylecgonine and (-)-ecgonine methyl ester inhibited BChE with Ki values of 15, 76 and 1300 microM, respectively. (+)-psi-Cocaine had Ki = 3 microM, p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (-)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar Ki values (160-170 microM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.