We have reinvestigated the hypothesis of relative importance of glomus cell E_m and _m to acute hypoxia by a non-invasive fluorescence micro-imaging technique using voltage sensitive dyes bis-oxonol and JC-1 respectively. Short- term (24h) cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E_m depolarization was indirectly confirmed by increase in bis-oxonol (an anionic probe) fluorescence due to graded increase in extracellular K⁺. Fluorescence responses of glomus cell E_m to acute hypoxia (P_OO2 10 Torr) indicated depolarization in 20%, being insensitive in 45% and hyperpolarization in 35% of the cells tested. Whereas, all PC-12 cells consistently depolarized to hypoxia. Further, glomus cell E_m hyperpolarization was confirmed with high CO (500 Torr). Glomus cell _m depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1µ M FCCP (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor) and acute hypoxia (P_OO2 10 Torr) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported as we named it, the metabo-membrane hypothesis of chemoreception.