Transgene expression may trigger intensive immune responses against heterologous protein resulting in shortening of gene expression and inflammation. This problem can be resolved by inoculating foreign protien antigen into embryos at an early development stage to induce immune tolerance. To induce immunological tolerance in laboratory chickens, human serum albumin (HSA) was inoculated into embryos by blood vessel microinjection (50 μg) at embryonation hours 65–67 or by yolk sac injection (100 μg) at embryonation days 3–7. All hatched chicks at 3-week-old were challenged with the same antigen four times at 10-day intervals, and serum samples were collected to determine anti-HSA antibody levels by enzyme-linked immunosorbent assay (ELISA). Chickens exposed to HSA at embryonation hours 65–67 by microinjection had 64.52％ incidence of tolerance. Immunological tolerance could be induced and maintained till 60-day-old by in ovo inoculation at embryonation day 6, and the formation of anti-HSA antibody could also be suppressed by inoculation at embryonation day 5 and 7 despite the shorter duration of tolerance. There was no significant effect of inoculating at embryonation day 3 and 4 on tolerance induction [ Acta Zoologica Sinica 51（5）：845 –851，2005].
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