An extraction and purification procedure, similar to that originally proposed by Silber et al. (11) for the fluorimetric determination of corticosterone in rat peripheral plasma, was adapted to quantitating 18-hydroxydeoxycorticosterone in arterial and adrenal venous rat plasma with the Porter- Silber reagent. 18-Hydroxydeoxycorticosterone was found to be the only 18-hydroxylated steroid present at a sufficiently high concentration in rat plasma to react with the reagent. The method can measure 1–5 μg of 18-hydroxydeoxycorticosterone in small (0.25–2.5 ml) or large (30–60 ml) volumes of adrenal vein and arterial blood plasma, respectively. Relative intensities of chromogens of standard cortisone and 18-hydroxydeoxycor.ticosterone varied little from one experiment to another over an extended period of time. Consequently,18-hydroxydeoxy corticosterone could be quantitated in terms of a cortisone standard by using the appropriate factor. Adrenal vein blood plasma from normal stressed rats had about 25 and 30 times as much corticosterone and 18-hydroxydeoxycorticosterone, respectively, as in the corresponding arterial blood plasma. The 18- hydroxydeoxycorticosterone plasma levels were approximately one half those of the corticosterone, whereas in control hypophysectomized or hypophysectomized ACTH-injected animals the levels were approximately the same. Administration of ACTH in hypophysectomized animals led to a 5- to 12-fold increase in 18-hydroxydeoxycorticosterone and corticosterone levels in adrenal vein blood plasma. 18-Hydroxydeoxycorticosterone is a natural steroid hormone biosynthesized in vivo by the rat adrenal gland, and its production, like that of corticosterone, is at least in part under ACTH control.