Truncated Bid (tBid) releases cytochrome c from mitochondria by inducing Bak (and Bax) pore formation in the outer membrane. An important issue is whether a second tBid action, independent of Bak and Bax, is also required to enhance cytochrome c mobility in the intermembrane spaces. To investigate this, we developed a kinetic analysis enabling changes in the diffusibility of cytochrome c in the intermembrane spaces of isolated mitochondria to be differentiated from changes resulting from Bak activation. Cytochrome c diffusibility in the intermembrane spaces was unaffected by changes in [tBid] over the range 0.5-19.0 pmol per mg of mitochondrial protein, when tBid-dependent Bak activation was increased several-thousand fold. However, high [tBid] (100 pmol mg(-1)) did increase diffusibility by approximately twofold. This was attributable to the permeability transition. Basal cytochrome c diffusibility in the intermembrane spaces in the absence of tBid was determined to be approximately 0.2 minute(-1), which is sufficient to support cytochrome c release with a half-time of 3.4 minutes. It is concluded that tBid has a monofunctional action at low concentrations and, more generally, that the basal cytochrome c diffusibility in the intermembrane spaces is adequate for rapid and complete cytochrome c release irrespective of the mode of outer membrane permeabilisation.