Several epidemiological studies have shown a positive association between dietary exposure to aflatoxin (AF) and an increased incidence of primary hepatocellular carcinoma (PHC). One area in which little information is available is the exposure of newborn children to AF in human breast milk. We report the development, validation and application of an enzyme-linked immunosorbent assay (ELISA) to the detection of AF in human breast milk. The assay allows the quantitation of 2 pg AFM1 per ml of milk using less than 10 ml of sample. A good correlation was observed between ELISA and an hplc-fluorescence technique using naturally contaminated milk at levels up to 40 pg AF per ml. Of 54 samples collected from women in rural villages in Zimbabwe, 6 were found to be positive (11%) in ELISA with levels up to 50 pg AF per ml. No positive samples were detected out of 42 milk samples obtained from women in France. This sensitive and rapid methodology will be useful in examining the importance of and interaction between exposure to AF and infection with hepatitis B virus (HBV) early in life.