Thiol reactivity was determined in rat heart mitochondria using chromophores of differing polarities: monobromobimane (MB), dithionitrobenzoate (Nbs 2), and bromobimane-q (MQ). The purpose of this study is to correlate reaction rates of protein thiols in the mitochondrial membrane with the oligomycin-inhibited and uncoupled states: In all cases investigated the reactivity of -SH groups toward MB decreases under the above conditions. In parallel with an increase of their uncoupling activities the uncouplers reduce the reaction rate of thiol groups toward Nbs 2 and, progressively, toward MQ, indicating differences in sensitivity of thiol groups to uncouplers depending on the polarity of the environment. The pattern of -SH reactivity under inhibition by oligomycin resembles that of carbonylcyanide- p-trifluoromethoxyphenylhydrazone. Functional changes of the mitochondrial membrane probably correlate with reactivity/polarity changes of membrane SH groups. Masking of membrane thiol groups thus is not specific for uncouplers but is also observed under inhibition with oligomycin.