A 3000 basepair (bp) region corresponding to the E3 region, the flanking pVIII and part of the fiber protein genes, of the two prototype strains (HNF-61 and HNF-70) of porcine adenovirus serotype five (PAdV-5) was sequenced. A potential E3 promoter and poly-A signals were identified. The size of the E3 region was 2039 (strain HNF-61) and 2020 bp (strain HNF-70) the largest E3 so far reported among PAdVs. Three open reading frames (ORF2-4) were identified within the E3 region. Based on the predicted amino acid (aa) sequences ORF2 was similar to other adenovirus E3 ORFs, ORF3 showed some similarity to a bovine adenovirus (BAdV-1) ORF. ORF4 was unique to PAdV-5. E3 mRNA transcripts were detected early in infection by Northern blot analysis. Genomic clones of HNF-70 with a 1505 or 1237 bp deletions in the E3 region were constructed to map non-essential regions. After transfection of the DNA into swine testicle cells, virions were recovered for only the shorter 1237 bp deletion. At least 60% of the E3 region was not essential for virus replication, bringing the theoretical vector capacity of a helper independent PAdV-5 to 2.9 kb.