We have demonstrated that the inhibition of nitric oxide synthase (NOS) in endothelial cells by either the NOS inhibitor, L-NMMA, or the internalization of calveolin-1 scaffolding domain, attenuated PAF induced increases in microvessel permeability (Am J Physiol 286: H195-H201, 2004) indicating the involvement of a nitric oxide (NO)-dependent signaling pathway. To investigate whether an increase in endothelial [Ca²⁺]_i is the initiating event and a calcium-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca²⁺]_i and NO production were measured in individually perfused rat mesenteric venular microvessels using fluorescent microscopy. When venular microvessels were exposed to PAF, endothelial [Ca²⁺]_i increased from 69 ± 8 nM to a peak value of 374 ± 26 nM within 3 min and then declined to a sustained level at 190 ± 12 nM after 15 min. The inhibition of NOS did not modify PAF-induced increases in endothelial [Ca²⁺]_i. PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increases in fluorescent intensity (FI), an indication of increased NO production, occurred in 75 ± 7% of endothelial cells in each vessel. The mean maximum increase in FI was 140 ± 7 % of the baseline. This increased FI was abolished by pretreatment of the vessel with L-NMMA, and attenuated in the absence of extracellular calcium. These results provided direct evidence in intact microvessels that increased endothelial [Ca²⁺]_i is the initial signal that activates eNOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.