We used density functional theory to investigate the capacity for carbon monoxide (CO) release of five newly synthesized manganese-containing CO-releasing molecules (CO-RMs), namely CORM-368 (1), CORM-401 (2), CORM-371 (3), CORM-409 (4), and CORM-313 (5). The results correctly discriminated good CO releasers (1 and 2) from a compound unable to release CO (5). The predicted MnCO bond dissociation energies were well correlated (R2≈0.9) with myoglobin (Mb) assay experiments, which quantified the formation of MbCO, and thus the amount of CO released by the CO-RMs. The nature of the MnCO bond was characterized by natural bond orbital (NBO) analysis. This allowed us to identify the key donor–acceptor interactions in the CO-RMs, and to evaluate the MnCO bond stabilization energies. According to the NBO calculations, the charge transfer is the major source of MnCO bond stabilization for this series. On the basis of the nature of the experimental buffers, we then analyzed the nucleophilic attack of putative ligands (L′=HPO42−, H2PO4−, H2O, and Cl−) at the metal vacant site through the ligand-exchange reaction energies. The analysis revealed that different L′-exchange reactions were spontaneous in all the CO-RMs. Finally, the calculated second dissociation energies could explain the stoichiometry obtained with the Mb assay experiments.