The N-terminal segment (residues 1-15) of hexokinase I (HKI) is essential for the binding of HKI to the outer membrane of the mitochondrion. Whether the N-terminal segment is merely a hydrophobic anchor to the membrane or has specific residues that are key determinants in the mitochondrion-HKI interaction is unclear. Recombinant wild-type HKI binds to mitochondria, but the removal of residues 1-15 abolishes such binding. Mutations A4L, A8L and Q5P individually cause a 10-fold decreases (relative to wild-type enzyme) in HKI binding to mitochondria. In contrast, mutations Q5A, Y10L and T12I decrease binding by approximately twofold. The mutations did not affect the catalytic properties of the enzyme, and all HKI constructs remained monomeric to concentrations as high as 10 micromolar. Results here are consistent with a helical conformation for the N-terminus of HKI, with residues A4 and A8 defining a contiguous surface that does not tolerate large hydrophobic side chains.