Martin et al., in 1990, proposed tri-enzyme treatments for food folate assay. We evaluated the effect of a-amylase and protease on folate contents after traditional folate conjugase (FC) treatment using four menus prepared for the determination of folate requirement in adult female volunteers. The menus were designed to be low in folate (32.3 pig/day, according to food folate tables). Food composites were homogenized in 0. l M phosphate buffer containing 1 % ascorbate and stored at -70°C. Protease (prepared from 5. griseus) digestions were performed for 16 hrs and a-amylase (purified from Bacillus species) for 4 hrs at 37°C followed by heating in boiling water for 10 min and chicken pancreas FC treatment (3 hrs). Folate was determined by J.. casei microbiological method. The entire assay procedure including extraction and enzyme treatments was repeated three times. Mean folate content in these daily menus was 56 μg after treatment with only FC. However, means were 90 μg with protease and FC (61 % increase), 126 μg with α-amylase and FC (125% increase), and 152 μg with a-amylase, protease and FC (171% increase). Our data indicate that the digestion with two enzymes in addition to FC treatment is essential to determine total food folate content. Previously published food folate contents may be grossly underestimated. Food folate tables should be revised using these enzyme treatments to accurately establish the dietary folate requirement of the human.