The distribution of P(i) between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J.111, 665-678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P(i) transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P(i) liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2-2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P(i) liberated was retained in the mitochondria. 2. Larger amounts of P(i) appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P(i) were identified: (a) a slow efflux of P(i) from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg(2+), partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P(i) and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P(i), is unaffected by uncoupling agents and is activated by endogenous or added Mg(2+). 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P(i) liberated was recovered in the suspending medium, unless inhibitors of P(i)-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg(2+) were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P(i) retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P(i) transporter provides the sole pathway for P(i) efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg(2+) on mersalyl inhibition of the P(i) transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15-17] and a postulated role of the adenine nucleotide exchange carrier in P(i) efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A.75, 4788-4792] are erroneous and should be discarded.
