Five purified carboxylesterases from rat liver microsomes show a differing capacity for the hydrolysis of ester- and amide-type drugs. The two closely related enzymes that are responsible for the microsomal hydrolysis of palmitoyl-CoA and long chain monoacylglycerides exhibit the highest propanidid-and aspirin-cleaving rates. The predominant nonspecific esterase of microsomes is responsible for the hydrolysis of procaine, clofibrate, isoarecaidine esters, butanilicaine, octanoylamide, and possibly butyryl thiocholine. Finally, the palmitoyl carnitine-cleaving esterase splits phenacetin and acetanilide. The purified nonspecific esterase with the lowest isoelectric point is not involved in the metabolism of the drugs mentioned.