The goal of this study was to characterize the effect of near-infrared light exposure on mitochondrial membrane potential, in vitro. We focused on the retinal pigmented epithelial (RPE) cells due to our interest in the visual health of military airmen exposed to infrared light, which causes thermal damage to the retina. Within RPE cells, an irradiance of 1.6 mW cm-2 for 30 minutes, resulting in a total fluence of 2.88 J cm-2, induces resistance to cell death in retinal pigmented epithelial cells exposed to a 1-sec hazardous pulse of 2 μm laser radiation 1. Thus, we examined the impact of this exposure on mitochondrial membrane potential in RPE cells. To do this, the fluorescent molecule, tetramethylrhodamine ethyl ester (TMRE), was used to quantify mitochondrial membrane potential. TMRE is a cell permeant, positively-charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Data from our study show that RPE cells exposed to an irradiance of 1.6 mW cm-2 for 30 minutes demonstrate elevations in mitochondrial membrane potential. This is the expectation if NIR light exposure is associated with oxygen consumption, as shown in previously published studies. Thus, by focusing on the uptake of TMRE in mitochondria, our findings provide additional details regarding the mechanism underlying the effect of NIR and potentially PBM in RPE cells. These findings may also apply to other cell types and red and NIR light exposures.