Automated 'in ovo' vaccination (IOJ) against Marek's disease in broiler chickens is now a routine practice in the poultry industry. This method deposits vaccine mostly into the extra-embryonic (EE) spaces around the embryo, principally the amnion. In earlier experiments of ours, manual EE (MEE) vaccine deposition has not produced good protection in contrast to the apparent efficacy of the automated 'in ovo' method. Two experiments are reported, one confirming this difference in protection and the other demonstrating that there are significant differences in the precise site of vaccine deposition between the two methods, with a higher incidence of allantoic deposition in the case of MEE (25.5%) compared to OIJ (5.7%). However, this finding alone cannot explain the difference in protection between the two methods. We suggest that different methods of vaccinal virus uptake from the amnion may be involved.