Knowledge of physiological mechanisms regulating vascular tone in human resistance arteries lags considerably that in animal vessels, mainly due to limited supply of non-pathophysiological human tissue. In myometrial arteries from biopsies obtained at term (≥ 37 weeks) Caesarean section of women with uncomplicated pregnancy, agonist-induced tone development arises partly through G protein-coupled Ca²⁺-sensitisation pathways (enhanced contractile response at a particular activating [Ca²⁺]_i).[1] As in prior animal studies, agonist responsiveness of myofilaments was examined at only one submaximal [Ca²⁺]_i, thus questioning the generality of the term agonist-mediated Ca²⁺-sensitisation. In this study we characterise Ca²⁺-sensitising actions of the thromboxane analogue U46619 in isometric, alpha-toxin permeabilised human myometrial arteries over a range of [Ca²⁺]_i (pCa 9 – 4.5). pCa was controlled by adjusting the ratio of K₂EGTA to CaEGTA in mock intracellular solution. A first cumulative, concentration-response (pCa-force) curve elicited concentration-dependent increases in tension, with mean ± S.E.M. maximal tension developed in pCa 4.5 (2.3 ± 0.4 kiloPascals, n = 7). A second successive curve in the same arteries elicited significantly lower tensions (P < 0.01, repeated measures (RM) 2 way ANOVA) with mean maximal response 0.6 ± 0.2 fold that in the first curve, but with similar EC₅₀ values determined from responses normalised to their own maximum force. Presence of 3.3 µM GTP, necessary for G protein-coupled, agonist-mediated signalling, did not alter pCa-force properties in first or second curves. In contrast to the reduced responsiveness of a second pCa-force curve, in arteries when the second curve was elicited in the presence of 1 µM U46619, there was significantly greater tension than that observed in the first curve without agonist in the same arteries (P < 0.01, RM 2 way ANOVA, n = 10). Mean maximal response in pCa 5.5 was 2.9 ± 3.3 fold that in the first curve, but mean EC₅₀ values were significantly lower (pCa 6.3 ± 0.1 vs. 6.6 ± 0.1, P < 0.01, Wilcoxan signed rank test); active tone was first elicted at pCa 7 compared with pCa 6.7 in the first curves. These second curve responses were also significantly greater than those in separate, time-matched arteries where second curves were elicited in the absence of U46619 (P < 0.01, 2 way ANOVA). A similar pattern of enhanced responses was observed in the presence of 0.1 µM endothelin. These findings indicate that agonist-induced Ca²⁺-sensitisation does indeed occur over a wide range of [Ca²⁺]_i further implicating this phenomenon as a contributor to contractile tone regulation in human arteries; this mechanism in myometrial arteries may participate in regulation of blood flow in the uteroplacental bed, important for matching fetal oxygen/nutrient demand to supply.