A method for embryo-blood collection (about 100 microliters/embryo) from chorioallantoic blood vessels of day 10 White Leghorn chick embryo was developed and the embryonic hemoglobin folate concentrations (folate/hemoglobin, microgram/g) were measured by the microbiological assay on microtitre dried blood spots to test the hypothesis whether homocysteine (HCY) would increase in folate depletion during early development stage of embryo. The results showed folic acid (FA) and HCY having been placed in the enlarged air cell could quickly pass through inner shell membrane and chorioallantoic membrane, and enter into blood, on the highly vascularized membrane of chick embryo aged 10 days. After 5 micrograms FA were placed on the inner shell membrane, there was a striking increase of hemoglobin folate with a peak in 60 min at 22.26 micrograms/g, followed by a decline. In contrast, 8 mumol of D. L-HCY did not result in the obvious increase of embryonic hemoglobin folate during the total transient time course (0-480 min). By 60 min posttreatment the hemoglobin folate concentration for joint group (HCY 8 mumol + FA 5 micrograms/embryo) had obviously decreased in comparison with single FA group (P < 0.001). The results supported the above hypothesis mentioned. Moreover, both advantages and disadvantages of this method were evaluated.