Endothelium derived relaxing factors (NO and/or EDHF) produce vasorelaxation: which could involve interaction between Ca signalling in endothelial cells and vascular smooth muscle cells (SMC). However, the mechanisms of reduction of [Ca²⁺]_i in SMC by NO and EDHF are still poorly understood. We have investigated possible roles of both NO-dependent and independent mechanisms in control of agonist induced Ca signalling in SMC of precapillary arterioles (PA) and small resistance arteries. A minimum of 17 vessels from 15 humanely killed rats were studied using confocal imaging of Fluo-4 loaded ureteric microvessels and mesentery arteries (MA) in situ. In the MA phenylephrine (PhE) produced complex Ca signalling consisting of asynchronous Ca waves and synchronous Ca oscillations. Both carbachol (CCh 10µM) and NO donors: SNP (20µM) and SNAP (20µM) inhibited this complex Ca signalling in pre-stimulated MA. Nifedipine (10µM) selectively blocked synchronous Ca oscillations and vasomotion but had no effect on asynchronous Ca waves. Nifedipine- resistant asynchronous Ca waves were abolished by ryanodine (10µM) or 2-APB (50µM). Carbachol and NO donors reversibly blocked PhE – induced Ca oscillations in the presence of nifedipine. L-NAME (100µM) selectively abolished the inhibitory effects of CCh on PhE induced Ca transients in the absence and the presence of nifedipine but not those induced by NO donors suggesting that NO plays a key role in inhibition of PhE induced Ca signalling in SMC under normal physiological conditions. Ryanodine selectively blocked asynchronous Ca waves in MA but enabled SMC to generate fast propagating Ca spikes (flashes) followed by sustained rise of Ca. Both the Ca spikes and sustained rise in Ca were blocked by nifedipine. Carbachol but not NO donors reversibly inhibited both fast and slow components of PhE induced Ca transients in the presence of ryanodine suggesting a little role of NO in these effects. In contrast to MA, in arterioles PhE induced exclusively Ca oscillations which were insensitive to ryanodine, nifedipine or removal of extracellular Ca but were inhibited by 2-APB. Neither CCh nor NO donors had any effects on PhE induced IP₃R mediated Ca oscillations in PA. Pretreatment of PA with TEA and BayK-8644 enabled them to respond to PhE with synchronous Ca spikes which were blocked by nifedipine but not 2-APB. These Ca spikes were reversibly blocked by CCh but not NO donors suggesting that NO-independent pathway is present in PA and can be physiologically active when the mechanisms of Ca entry via L-type Ca channels are activated. These data indicate marked differences in the mechanisms controlling Ca signalling evoked by agonists in SMC between macro- and microvessels and their modulation by endothelium derived relaxing factors.