Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly contagious upper respiratory tract infection in chickens. At present, ILTV vaccines are not satisfactory because of development of a latent carrier status in vaccinated birds. Development of recombinant virus vaccines has been hampered by the limited information available on the molecular level and organization of this virus. We isolated 3 assembly intermediates, designated A, B, and C from ILTV-infected cells. Analysis of [3H]thymidine- and [35S]methionine-labeled particles, and electron microscopic studies indicated that particle A was the empty capsid, particle B was the procapsid containing scaffolding protein, and particle C was the DNA-filled capsid. The ILTV procapsids could only be found in the nucleus, which indicated that procapsids could not translocate through the nuclear membrane until they packaged the DNA. The DNA-filled capsids migrated through the nuclear membrane and obtained an envelope from the inner membrane of the nucleus. The enveloped particles then migrated through the lumen of the endoplasmic reticulum into vacuoles in the cytoplasm. Infective virions were isolated from within the infected cells, indicating that budding through the cytoplasmic membrane is not a necessary step in ILTV maturation. Abundant arrays composed of tubules about 45 to 50 nm wide were found in the cytoplasm of chicken embryonic liver cells about 30 to 38 hours after infection. Comparison of the assembly intermediates and the DNA packaging pathway of ILTV with that of bacteriophage phi 29 indicates that similarity exists. A model for the pathway of ILTV assembly is proposed.