1-Aralkylimidazole-2-thiones have been shown to be potent multisubstrate inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1). In the present study, a series of 1-benzylimidazole-2-thiones was prepared to explore the effects of substitution in the benzyl ring on the inhibition of DBH. A detailed structure-activity relationship for in vitro activity was discovered and this was shown by a modified Hansch analysis to correlate (r = 0.91) with four key structural features of the benzyl ring: the presence of a hydroxyl at the 4-position, molar refractivity at the 3-, 4-, and 5-positions, inductive effects of the substituents at the 3-, 4-, and 5-positions, and pi-electron density. The affinity (Kis) of eight substituted inhibitors for DBH was shown to correlate (r = 0.75) with the affinity (KD) of comparably substituted tyramines for the ternary DBH-oxygen-tyramine complex. This correlate is used to support the hypothesis that binding of inhibitor to DBH occurs in a fashion that mimics the binding of tyramine substrates. The most potent inhibitors were selected for study in vivo in the spontaneously hypertensive rat model of hypertension. The changes in vascular dopamine and norepinephrine levels that resulted from oral administration of the inhibitors corresponded to the observed reduction in mean arterial blood pressure. A divergence between in vitro potency and in vivo efficacy upon oral dosing was noted and is suggested to result from an in vivo metabolic conjugation of the phenolic group of inhibitor.