Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injury following disease processes. However, this assumes that prolonged exposure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (EDL) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbital (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbenzimide (5 μg/ml; labels nuclei of all cells) and ethidium bromide (5 μg/ml; labels nuclei of injured cells) or had dyes topically applied 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. Noxious stimuli (i.e., hypoxia:FiO2 of 810% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were used to test the ability of ethidium bromide, when used in conjunction with intravital microscopy, to differentiate injured tissue. Video recordings at the surface of the EDL muscle were made every 30 min for 5 hr from which the number of perfused capillaries was counted (NCper). The numbers of bisbenzimide- and ethidium bromidelabeled nuclei were counted at the surface of the muscle and at two to three additional locations within the muscle (to a maximum depth of approximately 120-160 μm). The average NCper (19.05 ± 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 ± 125. The average number of ethidium bromide-labeled nuclei (380.4 ± 36.8) remained stable for at least 80-90 min, after which the number of labeled nuclei increased linearly. The number of ethidium bromide-labeled nuclei increased compared to control (P ≤ 0.05) following all noxious stimuli. Thus, within the first 80-90 min of exposure, use of fluorescent nuclear dyes in conjunction with intravital microscopy provides a means of evaluating spatial and temporal changes in tissue viability.