AbstractThe metabolism of [3H]labelled N‐Nitrosonornicotine a major constituent of the class of Tobacco Specific Nitrosamines was studied in adult and fetal human oesophageal cultures. The metabolites were separated by HPLC and were identified when compared to the standards as OH‐acid from 5′hydroxylation, NNN‐1‐N‐oxide formed via the pyridine N‐oxidation pathway and Keto acid from 2′hydroxylation in both the adult and fetal cultures. These results indicate that hydroxylation which leads to electrophilic diazohyroxides is the major pathway of NNN metabolism in cultured human oesophagus. In the adult cultures levels of metabolites formed were 20.32 pmoles/μg DNA of OH acid 11.08pmoles/μg DNA of NNN‐1‐N‐oxide and 7.67pmoles/μg DNA of Keto acid. In the fetal cultures levels were 10.85pmoles/μg DNA of OH acid, 9.40pmoles/μg DNA of NNN‐1‐N‐oxide and 7.91pmoles/μg DNA of Keto acid. These results indicate that α‐hydroxylation is the key step in the metabolic a