The subcellular distribution and sidedness on the membranes of four chemically and genetically distinct esterases (esterases ES-3, ES-4, ES-8, ES-15) in rat liver was investigated using selective substrates. (1) Rat liver homogenate was divided into nine subcellular fractions by differential centrifugation techniques. The cell fractions were assayed for the enzymatic hydrolysis of acetanilide (ES-3), propanidid, palmitoyl-CoA and monopalmitoylglycerol (ES-4), methyl butyrate and octanoylglycerol (ES-8), and decanoylcarnitine (ES-15). With all substrates, the highest specific activities were found in the rough and smooth endoplasmic reticulum fractions. This localization of the esterases was confirmed by labelling the cell fractions with the specific, covalently binding inhibitor bis(4-nitro[14C]phenyl) phosphate. The enzymatic hydrolysis of the palmitoyl esters in differing cell fractions did not completely parallel that of propanidid. This confirms the well-known existence of palmitoyl-CoA hydrolases other than esterase ES-4. (2) Density gradient fractionations with crude mitochondria indicated that a low amount of at least one of these carboxylesterases was an integral part of these organelles too. (3) Proteinase treatment reduced the non-specific esterase activities as well as lipase activities versus dioctanoylglycerol, acylcarnitines and palmitoyl-CoA only in detergent-disrupted microsomal vesicles. This might indicate a lumenal orientation of these enzymes. However, of the charged substrates palmitoylcarnitine and palmitoyl-CoA only the latter one showed the typical latency to be expected for a hydrolysis in the lumen of the endoplasmic reticulum.