The inhibitory effect of three SH reagents, mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide, on Pi transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (Kd less than 10 microM) and low affinity to mitochondria. Inhibition of Pi transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5-4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 microM, inhibition of Pi transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 microM, the rate of reaction with the Pi carrier is considerably decreased. At a concentration of 100 microM, 5,5'-dithio-bis-nitrobenzoate fully inhibits Pi transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40-50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the Pi carrier, begin to react. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5'-dithio-bis-nitrobenzoate, and N-ethylmaleimide with the Pi carrier. Preincubation with Pi has a similar effect. We propose that both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and Pi act by increasing the acidity of the mitochondrial matrix. Protonation of the Pi carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the Pi carrier.