The cytoprotective properties of carbon monoxide (CO) gas and CO‐releasing molecules (CORMs) are well established. Despite promising pre‐clinical results, little attention has been paid to the toxicological profile of CORMs. The effects of CORM‐2 and its CO‐depleted molecule (iCORM‐2) (20–400 μM) were compared in primary rat cardiomyocytes and two cell lines [human embryonic kidney (HeK) and Madine‐Darby canine kidney Cells (MDCK)]. Cells were assessed for cell viability, apoptosis, necrosis, cytology, mitochondrial energetics, oxidative stress and cell cycle arrest markers. In separate experiments, the anti‐apoptotic effects of CORM‐2 and i‐CORM‐2 treatment were compared against CO gas treatment in HeK and MDCK lines. H₂O₂‐induced cellular damage, measured by lactate dehydrogenase (LDH) release from primary cardiomyocytes, was reduced by 20 μM CORM‐2; LDH activity, however, was directly inhibited by 400 μM CORM‐2. Both CORM‐2/iCORM‐2 and CO gas decreased cisplatin‐induced caspase‐3 activity in MDCK and HeK cells suggesting an anti‐apoptotic effect. Conversely, both CORM‐2 and iCORM‐2 induced significant cellular toxicity in the form of decreased cell viability, abnormal cell cytology, increased apoptosis and necrosis, cell cycle arrest and reduced mitochondrial enzyme activity. Comparison of these markers after CO gas administration to MDCK cells found significantly less cellular toxicity than in 100 μM CORM‐2/iCORM‐2‐treated cells. CO gas did not have an adverse effect on mitochondrial energetics and integrity. Release of CO by low concentrations of intact CORM‐2 molecules provides cytoprotective effects. These results show, however, that the ruthenium‐based CORM by‐product, iCORM‐2, is cytotoxic and suggest that the accumulation of iCORM‐2 would seriously limit any clinical application of the ruthenium‐based CORMs.