A mutation in codon 249 of the TP53 gene (249(Ser)), related to aflatoxin B(1) exposure, has previously been associated with hepatocellular carcinoma risk. Using a novel internal standard plasmid, plasma concentrations of 249(Ser)-mutated DNA were quantified by electrospray ionization mass spectrometry in 89 hepatocellular carcinoma cases, 42 cirrhotic patients, and 131 nonliver diseased control subjects, all from highly aflatoxin-exposed regions of The Gambia. The hepatocellular carcinoma cases had higher median plasma concentrations of 249(Ser) (2,800 copies/mL; interquartile range: 500-11,000) compared with either cirrhotic (500 copies/mL; interquartile range: 500-2,600) or control subjects (500 copies/mL; interquartile range: 500-2,000; P < 0.05). About half (52%) of the hepatocellular carcinoma cases had >2,500 copies of 249(Ser)/mL plasma, corresponding to the prevalence of this mutation in liver tumors in The Gambia. In comparison, only 15% of control group and 26% of cirrhotic participants exceeded this level (P < 0.05). Further subset analysis revealed a statistically significant, quantitative relation between diagnosis of hepatocellular carcinoma and levels of 249(Ser) detected at 2,501 to 10,000 copies/mL plasma (odds ratio, 3.8; 95% confidence interval, 1.3-10.9) and at >10,000 copies/mL plasma (odds ratio, 62; 95% confidence interval, 4.7-820) when compared with control subjects and after adjusting for age, gender, recruitment site, hepatitis B and C serologic status, and total DNA concentration. Levels of >10,000 copies of 249(Ser)/mL plasma were also significantly associated with the diagnosis of hepatocellular carcinoma (odds ratio, 15; 95% confidence interval, 1.6-140) when compared with cirrhotic patients. Potential applications for the quantification of 249(Ser) DNA in plasma include estimation of long-term, cumulative aflatoxin exposure and selection of appropriate high-risk individuals for targeted intervention.