We have recently shown that oxidant activated Protein Kinase G (PKG) contributes to small artery contractility by regulation of the Ca2+ spark/Large conductance Ca2+-activated K+ (BK) channel axis. To explore characteristics of this signalling pathway in more detail, age-matched (12-week-old) male wild type C57BL/6J (WT) and PKG[Cys42Ser]KI (KI) mice were used, in which PKG is resistant to oxidant induced dimerization. Mice were euthanized by cervical dislocation. Vascular function of mesenteric resistance arteries was examined in vitro with pressure myography and wire myography. Ca2+ sparks and caffeine-induced Ca2+ transients (indicative of sarcoplasmic reticulum (SR) Ca2+ load) were imaged with high-speed spinning-disc confocal microscopy. Western blot protocols examined ryanodine phosphorylation. In WT arteries, Ca2+ spark frequency increased with intraluminal pressure but reached a ‘Ca2+ spark frequency-ceiling’ at 80 mmHg (n=19 arteries from 8 mice). Ca2+ spark frequency was significantly increased at 80 mmHg in WT (from 8.3±1.3 Hz to 23.6±2 Hz; n=9 arteries from 4 mice; P=0.0045) but not in the KI arteries (from 5.76±1.6 Hz to 6.9±0.9 Hz; n=9 arteries from 3 mice). Similarly, exogenous H2O2 increased Ca2+ spark frequency in WT arteries at low pressures (n=7 arteries from 3 mice) but did not have this effect at higher intraluminal pressures (n=8 arteries from 3 mice). Conversely, caffeine-induced Ca2+ transients showed very little relationship with intraluminal pressure, although at higher pressures SR Ca2+ content was reduced in WT (n=7 arteries from 4 mice) compared with KI arteries (n=6 arteries from 4 mice, P<0.001 between WT and KI arteries at 110 mmHg). Both exogenous H2O2 and the BK agonist NS11021 vasodilated pressure-constricted mesenteric arteries equivalently (WT: 99±1.6%, n=7 arteries from 5 mice; KI: 97±4%, n=7 arteries from 6 mice in the presence of 30 µM H2O2 and WT: 95.3±2.4% vasodilation n=3 arteries from 3 mice; KI: 93.9±1% vasodilation n=3 arteries from 2 mice at 3 µM NS11021). 30 µM H2O2-induced vasodilation of WT arteries mounted in a wire myograph (67.3±11.9%, n=8 arteries from 7 mice) but not the PKG[C42S]KI (7.3±3%, n=5 arteries from 4 mice). H2O2 caused phosphorylation of the ryanodine receptor at S2808 from freshly dissected, pooled (unpressurized) mesenteric arteries with or without incubation with 30 μM H2O2 (P=0.0426 between WT and WT+ 30 µM H2O2, n=4 arteries from 4 mice, respectively). Oxidant activated PKG appears to target the ryanodine receptor to increase Ca2+ spark frequency and maintain BK function during pressure induced constriction. However, once activated, further increases of pressure or exogenous oxidants cannot additionally increase the activity of this vasodilatory pathway, despite only low-level activity of the BK channel.