Mitochondrial function is vital for many physiological processes, and the importance of motility, position and morphology of these organelles is starting to be appreciated. Alterations in mitochondrial position affect localised cellular events, changes in mitochondrial morphology influence metabolic responses, and the motility of mitochondria adapts to and regulates different cellular states. Quantification of the shape and motility of mitochondria is challenging due to their small dimensions, rapid velocities and crowded cellular environments. We developed live-cell image analysis tools which have revealed changes in mitochondrial dimensions, position and motility in vascular smooth muscle that occur with age, hypertension and proliferation. Flicker-assisted Localisation Microscopy (FaLM) discriminates the shape and relative position of individual mitochondria by measuring the local covariance of potentiometric fluorophore intensity changes during stochastic “flickers” of mitochondrial membrane potential (ΔΨm). Freshly-isolated rat cerebral artery myocytes loaded with TMRE were imaged for a sufficient duration (10-20 min) to record spontaneous ΔΨm flickers. FaLM revealed that in a rat model of hypertension mitochondria were larger (SHR, mitochondrial area 0.83±1.97 μm2 cf. 0.35±3.08 μm2 in normotensive WKY; n=5 animals each, p<0.01; all values are mean±SEM, compared by two-sample Kolmogorov-Smirnov or t-test), occupied a greater proportion of the cell (19.7±1.2% in SHR cf. 7.0±1.4% in WKY; n=5 animals each, p<0.01) and were clustered more tightly (1.9±0.04 μm between centres of neighbouring mitochondria in SHR cf. 2.2±0.06 μm in WKY; n=5 animals, p<0.01). Mitochondria were also found to be larger in myocytes from aged cf. younger rats (1.81±0.05 μm2 mitochondrial area in 18 month-old cf. 0.35±0.02 μm2 in 3 month-old; n=5 animals each, p<0.01), again occupying a greater proportion of the cell (18.1±5.2% in 18 month cf. 8.5±3.2% in 3 month; n=5, p<0.01). A notable proportion of mitochondria in aged animals were highly-elongated (length:width ratio>3; 4.3% of mitochondria in 18 month cf. 0.4% in 3 month; n=5 animals, p<0.01). Mitochondria in younger animals were often motile, whereas those in aged animals were not (19% of mitochondria in 3 month cf. 0.12% in 18 month; n=5 animals, p<0.01). We have previously shown that in native cerebral artery myocytes (from 10-15 week-old guinea pigs) mitochondria were not motile but that a switch to extensive motility was involved in – and necessary for – serum induced proliferation, with bursts of motion as fast as 1 µm/s tracked by our MotionStudio analysis (1,2). Thus, mitochondrial motility within smooth muscle decreases with age but may restart during cellular remodelling to a proliferative phenotype. Concurrent with decreased motility with age, mitochondrial size increases – a change that also occurs in hypertension.