FRET (Forster resonance energy transfer)-based sensors are powerful tools for understanding physiological mechanisms at the molecular level. Here, we used transgenic mice that express, specifically in smooth muscle, a FRET-based, exogenous myosin light chain kinase biosensor (‘exMLCK’) to observe for the first time changes in [Ca2+/Calmodulin] and MLCK activation within arteries in vivo (‘intra-vital FRET imaging’). exMLCK biosensor mice were anesthetized (1.5% isoflurane) and placed on the stage of a ‘macro’ epi-fluorescence microscope (Olympus MVX 10) equipped with an image splitter and a digital CCD camera. Arterial blood pressure (AP) was recorded. Femoral or mesenteric arteries were exposed through cutaneous incisions. Quantification of exMLCK FRET in vivorequires precise assessment and removal of tissue intrinsic fluorescence and extraneous sources of light. Intrinisic fluorescence (of isolated arteries) was negligible in comparison to exMLCK fluorescence. In vivo, background fluorescence was minimized by dissection of surrounding tissues and the use of an appropriate field diaphragm. Artery diameter was determined using edge detection and spatially averaged exMLCK FRET ratio (CFP/YFP) was determined from regions of interest. In the basal state (mean AP of ∼90 mm Hg), average exMLCK FRET ratio in femoral artery walls was 1.8 to 1.9, similar to that obtained by us in isolated arteries. Application of the α1-adrenoceptor agonist, phenylephrine (PE) directly to an artery increased exMLCK FRET ratio transiently to a peak of about 2.2 and caused a local vasoconstriction of ∼33%, without changing AP. Intra-venous application of PE elevated AP and caused smaller changes in exMLCK FRET ratio and artery diameter (than direct application). In conclusion, quantitative intra-vital FRET imaging in arteries of transgenic animals is feasible and will permit observation of specific molecular events in tissues of living animals. (AHA, NIH-HL078870).