ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH-treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant 2 Na,K-ATPase subunits (2^R/R). Since tail-cuff measurements involve restraint-stress, the present study used telemetry to distinguish between an effect of ACTH on resting blood pressure versus an ACTH-enhanced stress-response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant 2^R/R mice versus wild type mice (ouabain-sensitive 2 Na,K-ATPase, 2^S/S). Pretreatment BP was not different between the two genotypes, but following 5 days of ACTH-treatment, BP increased in 2^S/S (104.0±2.6 to 117.7±3.0 mmHg), but not in 2^R/R (108.2±3.2 to 111.5± 4.0 mmHg). To test the hypothesis that ACTH-induced hypertension is related to inhibition of 2 Na,K-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, BP and regional blood flow were measured. Results suggest that there is differential sensitivity of renal, mesenteric and cerebral circulations to ACTH, and that the response depends on the ouabain-sensitivity of the 2 Na,K-ATPase. Baseline cardiac performance was elevated in 2^S/S but not 2R/R mice. Overall, the data establish that the 2 Na,K-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism of hypertension appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations,presumabaly caused by elevated levels of circulating cardiotonic steroids.