Vectors derived from canine adenovirus type 2 (CAV-2) are attractive candidates for gene therapy and live recombinant vaccines. CAV-2 vectors described thus far have been generated by modifying the virus genome, most notably early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous descriptions of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study, we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We examined the major late transcription unit (MLTU) and determined its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader (224 nt) at their 5' end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence yielded a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions that were deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimization.