Using a competitive enzyme immunoassay, one or more substances recognized by a monoclonal antibody against aflatoxin B₁ were detected in human urine samples collected in Denmark. The concentration of urinary aflatoxin-like substances was equivalent to 0.0-6.5 ng aflatoxin B₁/mg creatinine. A truly competitive interaction in the immunoassay was found between aflatoxin-like substances and aflatoxin B₁. Aflatoxin-like substances could be isolated in small quantities from urine by affinity chromatography. The quantity of urinary aflatoxin-like compounds in the samples collected showed a skewed normal distribution (80 individuals). In order to explain the seemingly high level of aflatoxin-like material in urine samples from people living in a cold temperate climate, the source of aflatoxin-like compounds was investigated. In a dietary restriction study, potential dietary factors leading to excretion of aflatoxin-like compounds were investigated. Our data indicate that the excretion of these compounds by healthy Danes depends mainly on the food ingested 24-48 hr before urine samples were collected. In particular, the excretion of aflatoxin-like substances was increased when diets include beer, dairy products or meat. A map of the epitope recognized by the antibody was constructed from the results of competition studies with several AFB₁ analogues. The epitope map was used to draw chemical structures representing the minimal requirements for antibody recognition. An on-line search was conducted among the 98.2 × 10⁶ structures in the Chemical Abstracts and Registry Databases (STN, Columbus, OH) and provided strong evidence that only aflatoxins or aflatoxin derivatives are recognized by the antibody. The possible chemical structures of the aflatoxin-like substances are discussed.