Replication of a recombinant herpesvirus of turkey vaccine expressing infectious laryngotracheitis virus genes (rHVT-LT) was evaluated in specific pathogen free (SPF) and commercial broiler chickens after various vaccination protocols (amniotic route at embryonation day [ED] 18; intra-embryonic route at ED 19; and subcutaneous at 1 day of age [s.c.]). Three experiments were conducted: in the first experiment, replication of rHVT-LT vaccine was chronologically evaluated and compared with the replication of herpesvirus of turkey (HVT) in SPF chickens; in the second experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in SPF chickens; and in the third experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in commercial broiler chickens with maternal antibodies against HVT and infectious laryngotracheitis virus (LTV). rHVT-LT vaccine replicated in chickens after in ovo (ED 18 and ED 19) or s.c. administration at a similar level. In vivo replication of rHVT-LT vaccine was slower than HVT vaccine. However, in vivo both rHVT-LT and HVT vaccines replicated at similar levels. Both vaccines were consistently detected in the spleen and feather pulp and at lower frequency in the lung. The frequency of samples with detectable levels of rHVT-LT DNA was lower in broiler chickens than in SPF chickens, probably due to interactions with maternal antibodies. Differences between SPF chickens and broiler chickens were found also in the transcription of the LTV glycoprotein I gene (gI). In SPF chickens, in ovo inoculation resulted in a higher number of spleen samples with detectable gI transcripts than s.c. inoculation. In broiler chickens, however, no differences in the level of gI transcripts in spleen samples were found between chickens vaccinated in ovo and those vaccinated by the s.c. route. Transcription of LTV gI gene in lung samples was very low in both SPF and broiler chickens. Further studies to evaluate the mucosal immune response elicited by rHVT-LT in chicken with and without maternal antibodies are warranted.