There are two competing models for ligand entry and exit in globins. In the histidine gate model, a channel from the heme iron to the solvent opens by the outward rotation of the His(E7) side chain (7 th residue of the E-helix). In the multiple paths model, ligands diffuse through the protein matrix and exit at multiple points at its surface. Previous workers solved crystal structures to identify channels in myoglobin (Mb) with n-alkyl isocyanides (CNRs), a long flexible ligand that acts as a molecular "Ariadne's thread." When bound to Mb, a CNR points either toward solvent through an opened His(E7) (out conformation) or into the back of the distal pocket (in conformation). To measure the in/out equilibrium in solution, FTIR spectra have been collected for wild-type and mutant MbCNRs. The fraction of CNRs that point in (Fin) is regulated by the distal pocket volume, the freedom of the His(E7) side chain to rotate outward, and an unfavorable hydrophobic effect for CNRs that point outward into solvent. The relative importance of distal histidine flexibility and pocket volume on diatomic ligand binding with Mb has been assessed by correlating F in for bound CNRs with O2 and NO binding parameters. These correlations indicate strongly that: (1) CNRs and diatomic ligands use the histidine gate; (2) the volume of the binding pocket regulates non-covalent ligand capture and covalent bond formation with the iron atom; (3) the "baseball glove" model of ligand binding to Mb applies for all ligands; and (4) CNRs serve as useful transition state analogs for the diatomic ligand binding reactions of all globins.