To identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1B small , or E1B large protein. Sera against E1A, E1B small , and E1B large immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1B small , DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1B small and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1B small , and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1B large coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1B small is not essential for replication of PAV-3, and (c) E1B large is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene.