We investigated why liver mitochondria from small mammals are leakier to protons than those from larger mammals. Sixty-nine percent (+/-23%) of the proton leak differences appeared to relate to membrane area (less inner membrane surface area in larger animals); any residual differences must reflect differences in membrane properties. There were differences in phospholipid fatty acid composition; unsaturation index, monounsaturates, palmitate (16:0), stearate (18:0), docosahexaenoate [22:6(n-3)], and the 22:6(n-3)/22:5(n-3) ratio all correlated with body mass. Proton flux per square centimeter did not correlate significantly with body mass or, in general, with phospholipid fatty acid composition, suggesting little role for fatty acid composition in determining proton leak in mammals of different body mass. However, unsaturation index and n-3 polyunsaturated fatty acid content correlated significantly with proton leak per milligram phospholipid when literature data from reptiles and rats in different thyroid states were included, giving some support to suggestions of a general role for phospholipid fatty acid composition in determining mitochondrial proton leak.