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The mitochondrial electrical membrane potential (Δ
ψ
) is the main component of the proton motive force (Δ
p
) generated across the inner mitochondrial membrane during electron flow through the respiratory chain. Among the techniques available to assess Δ
ψ
, methods that rely on the spectrophotofluorometric responses of dyes are widely employed for whole suspensions of isolated mitochondria or permeabilized cells. Safranine is one of the dyes currently used most often for this purpose. Safranine is a lipophilic cationic dye that undergoes optical shifts upon its potential-dependent distribution between the external medium and the intramitochondrial compartment and on its stacking to inner mitochondrial membrane anionic sites. The association between the optical changes of safranine and the membrane potential allows unknown Δ
ψ
values to be estimated from an equation describing their relationship. Here, we describe the use of safranine as a fluorescent indicator of Δ
ψ
in isolated mitochondria and digitonin-permeabilized cells. We present suitable conditions to employ safranine as a Δ
ψ
indicator.
