BACKGROUND: Recreational use of nitrous oxide (NO) leads to neurological disorders including combined subacute degeneration of spinal cord, psychological disorders, and thrombosis. Serum or urine NO assays could not be routinely performed. Hence, it is necessary to investigate other biological markers such as metabolic markers. We aimed here to challenge the three main biological markers used for the diagnosis of nitrous oxide abuse as total vitamin B12, homocysteine, and methylmalonic acid. METHODS: We retrospectively collected clinical and biological data from 52 patients with known, documented chronic NO abuse and associated clinical signs (peripheral neuropathy disability score or thrombosis event). Sera and plasma total vitamin B12, methylmalonic acid (MMA), and homocysteine were performed to identify the most specific marker of chronic NO intoxication and related clinical outcomes. RESULTS: Plasma homocysteine was almost consistently increased in case of NO chronic consumption, whereas MMA increase and total vitamin B12 decrease are not systematically found. Our results showed that none of the markers are correlated with levels of NO consumptions. However, homocysteine and MMA are correlated with clinical severity, but MMA seems to be a better marker of clinical severity. CONCLUSION: There is no specific marker of nitrous oxide abuse according to levels of consumption, total vitamin B12 decrease could not be used either as consumption or as severity marker. However, we showed that homocysteine is consistently increased and could be used as marker of recent NO consumption. On the other hand, we showed that MMA could be used as a marker of clinical gravity.