New synthetic substrates for serum pseudo-cholinesterase activity were compared with the common substrates for the routine assays, with regard to reactivity, specificity and stability; o- and m-toluoylcholine as well as o- and m-toluoyldimethylaminoethanol esters had selective specificities for pseudo-cholinesterase. The last three substrates, however, were unstable in solution at 4 degrees C. On the other hand, o-toluoylcholine could be stored in solution for several days with no appreciable degradation, and it was extremely stable with regard to pH and temperature. No or little hydrolysis of o-toluoylcholine was observed by various enzymes other than pseudo-cholinesterase. The enzymatic method using o-toluoylcholine as substrate was reproducible, and the results correlated well with those obtained using butylthiocholine as substrate and 5,5'-dithiobis-(2-nitrobenzoic acid) as color reagent. In conclusion, o-toluoylcholine is a favorable substrate for the determination of serum pseudo-cholinesterase activity.