SUMMARY. The Arkansas-Delmarva poultry industry (ArkDPI) infectious bronchitis virus (IBV) vaccine is effective when administered by eye drop, where the vaccine virus is able to infect and replicate well in birds and is able to induce protection against homologous challenge. However, accumulating evidence indicates that ArkDPI vaccine is ineffective when applied by hatchery spray cabinet using the same manufacturers' recommended dose per bird. For this study, we aimed to determine the minimum infectious dose for spray-administered ArkDPI vaccine, which we designate as the dose which achieves the same level of infection and replication as eye drop-administered ArkDPI vaccine. To this end, we used increasing doses of commercial ArkDPI vaccine to vaccinate 100 broiler chicks at day of hatch, using a commercial hatchery spray cabinet. The choanal cleft of each bird was swabbed at 7 and 10 days post-vaccination and real time RT-PCR was performed. We observed that the level of infection and replication with spray vaccination matches with that of eye drop vaccination when chicks received 100 times the standard dose for the commercial ArkDPI vaccine. We further examined the S1 spike gene sequence from a subset of re-isolated ArkDPI vaccine virus samples and observed that certain nucleotide changes arise in vaccine viruses re-isolated from chicks, as previously reported (10, 12, 18). This suggests that the ArkDPI vaccine has a certain virus subpopulation that, while successful at infecting and replicating in chicks, represents only a minor virus subpopulation in the original vaccine. Thus, the minimum infectious dose for the ArkDPI vaccine using a hatchery spray cabinet appears to be dependent on the amount of this minor subpopulation reaching the chicks.