We have shown that cytochrome c (cyt c) diffuses primarily in three dimensions in the intermembrane space (IMS) of intact mitochondria at physiological ionic strength (I). Recently, we found that a small percentage (11.2 +/- 2.1%) of endogenous cyt c remains bound to inner mitochondrial membranes (IMM) at high, physiological I (I = 150 mM), even after extensive washing with solutions at physiological I, overnight dialysis, changes in medium osmolarity, or further purification of IMM at high I using self-generating Percoll gradients. Measurements of heme c/heme a ratios, and electron transport (ET) reactions in which cyt c participates, confirmed the presence of a low amount of this I-resistant, membrane-bound form of cyt c (MB-cyt c), that had one third of the ET activity of electrostatically-bound cyt c (EB-cyt c), and which could not account for maximal ET rates. The amount of MB-cyt c was significantly increased above endogenous MB-cyt c by exposing KCl-washed IMM to increasing concentrations of exogenous cyt c. Also, subjecting large unilamellar vesicles (LUV) to successive cycles of cyt c binding/high I KCl-washes gave progressive increases in MB-cyt c. These protocols allowed in vitro characterization of MB-cyt c. The I at which binding takes place affects the affinity of cyt c for membranes, and oxidized cyt c had a greater intrinsic affinity for IMM or SUV than reduced cyt c. MB-cyt c appears to be bound partially by hydrophobic interactions since MB-cyt c was detected on negatively charged (asolectin) LUV and also on neutral, zwitterionic (phosphatidylcholine) LUV at high I. Consistent with the concentration-dependent changes in MB-cyt c, decreasing the IMS-volume of intact mitochondria (i.e., increasing th endogenous IMS-cyt c concentration) by metabolic or osmotic means increased the amount of MB-cyt c. After cyt c was delivered into the IMS by liposome-mediated low pH-induced fusion, resonance energy transfer showed a time-dependent cyt c-membrane proximity which was consistent with slow exchange of soluble IMS-entrapped cyt c molecules with a population bound to membranes at I = 150 mM. We conclude that, even though the majority of functional IMS-cyt c diffuses in three dimensions, a small portion remains firmly bound on the surface of the IMM under I conditions that are physiological for intact mitochondria. The occurrence of MB-cyt c may reflect an intrinsic conformational flexibility in cyt c, that allows a degree of membrane penetration and the formation of hydrophobic interactions which stabilize the membrane-bound form. The persistence of cyt c-membrane interactions under physiological I conditions indicates that cyt c-mediated ET in the IMS involves both fast (3D-diffusion) and slow (2D-diffusion) pathways for electron transfer.