This paper is concerned with some physiologic relationships of non-specific esterases of tissues of the rat, dog, and man which were investigated by a colorimetric technique. Two patterns of hydrolysis were revealed that were characteristic of several tissues. I t was found also that the esterase of rat testis is produced in the interstitial cells, that its content is under control of the hypophysis, and that its concentration is related to the production of androgen by the testis.The colorimetric method employed chromogenic substrates, colorless compounds that liberate color on hydrolysis. The color value is read directly, the depth of color produced by enzymatic hydrolysis being directly related to activity of the enzyme. The chromogenic substrate techniques of enzymatic investigation combine accuracy, delicacy, and simplicity. All the chromogenic substrates, beginning with the first use of this technique by Ohmori (1), have been compounds of phenolic acid-base indicators esterified with phosphoric (1, 2), glucuronic (3), sulfuric (4), or fatty acids (5). In the present studies the substrates were the aeyl esters of p-nitrophenol previously synthesized in this laboratory.Three principal esterase activities in animal tissues can be differentiated according to their distribution, their efficiency against various substrates, and the effect of inhibitors (5, 24) upon them. An enzyme cannot be characterized with certainty until it has been isolated in a pure state and this has not been achieved with any of the esterases. However, from indirect evidence esterolysis seems to be brought about by three separate types of enzymes; in this paper the effects will be designated according to conventional usage as cholinesterase, non-specific esterase, and lipase. No doubt there is some overlapping of their activities and, as will be brought out, the chief differentiating characteristics are quantitative rather than qualitative.The choline esters are sufficiently soluble in water to serve as satisfactory substrates in vitro. Cholinesterase has an entirely different distribution than the other esterases.Maruay and Nachmansohn (6) found that the cerebral cortex and uterus of guinea pig hydrolyzed acetylcholine more effectively than extracts of kidney or liver did. In swine (7) aeetylcholine is hydrolyzed more rapidly by certain structures which, in descending order of magnitude, are the parotid, lachrymal and sublingual glands, Fallopian tube, jejunal and gastric mucosa, and the medulla oblongata. Mendel,
