Folate contents in samples obtained from cow's milk and breast milk were determined by a microbiological assay method with Lactobacillus casei ATCC 7469 as a test organism. For preparation of sample solution from each material, an improved technique by pretreatment with protease was employed throughout this study. Free and total folate contents in processed milk and breast milk estimated by the improved method amounted to 120〜190% of those obtained by ordinary method. After heating at 100℃ for 5 min, however, free folate activities in cow's milk decreased in some degree. Total folate content in farm milk was 115.1±28.1 μg/l, that of sterilized milk was 118.4±53.2 μg/l, and 115.8±45.3 μg/l in processed milk. These results strongly suggest that cow's milk, even though fresh or processed, have an almost the same folate level and a very wide range in the concentration of the vitamin. Total folate activity in breast milk was 71.8±26.1 μg/l, indicating that it has also a wide range of folate concentration. A favourable breast m,ilk with a high folate level was obtained from healthy women during lactation.