Background: O2 pathways in animal hemoglobins and myoglobins are controversial. Results: Ligands enter and exit sperm whale Mb and Cerebratulus lacteus Hb by completely different pathways. Conclusion: Rational mutagenesis mapping can identify ligand migration pathways and provides experimental benchmarks for testing molecular dynamics simulations. Significance: Globins can use either a polar gate or an apolar tunnel for ligand entry.