Toombak is a type of snuff used extensively in the Northern Sudan by a virtually non-smoking/nondrinking population. This Sudanese snuff contains inordinately high levels of the tobacco-specific nitrosamines (TSNAs) Nl-nitrosonornicotine (NNN) and (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These are considered to be major contributors to the induction of cancers of the aerodigestive tract in tobacco chewers, snuff dippers, and smokers. To determine if toombak use may be associated with the induction of mutations in the p53 tumor suppressor gene, we screened four head and neck squamous cell carcinomas (SCCs) obtained from three toombak-using patients and one non-toombak-using patient using polymerase chain reaction/single-stranded conformational polymorphism analysis and DNA sequencing. p53 mutations were found in tumors resected from two of three toombak-using patients, one at codon 282 (CGGarg-->TGGtrp) and the other in intron 6 (AT-->GC). No p53 mutations were observed in the tumor from the non-toombak-using patient. The observed mutations were similar in spectrum to those induced by nitrosamines in mouse lung tumors. No K-ras (codons 12 and 13) or H-ras (codon 12) mutations were found in any of the tumors. These results suggest that toombak components such as TSNAs may induce p53 mutations in head and neck SCCs and are likely contributors to the tobacco-induced carcinogenic load in humans.