The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.