Porcine adenovirus has been proposed as a potential vector for generating novel and effective vaccines for pigs. As a prerequisite for the generation of helper-dependent porcine adenovirus-3 (PAV-3) vectors, two E1-complementing porcine cell lines expressing E1 proteins of human adenovirus-5 (HAV-5) were made. These cell lines could be efficiently transfected with DNA and allowed the rescue and propagation of a PAV-3 recombinant, PAV201, containing a 0.597 kb E3 deletion and a 0.803 kb E1A deletion. Our data demonstrate that E1A proteins of HAV-5 have the capacity to transform foetal porcine retina cells and complement for the E1A proteins of PAV-3. The green fluorescent protein (GFP) gene placed under the control of a cytomegalovirus immediate early promoter was inserted into the E1A region of the PAV201 genome. Using these cell lines, a helper-dependent PAV-3 recombinant expressing GFP, PAV202, was constructed and characterized. The wild-type PAV-3 and the recombinant PAV202 expressing GFP were used to determine the ability of the virus to enter and replicate in cells of human and animal origin under cell culture conditions. Our results suggest that PAV-3 enters but does not replicate in dog, sheep, bovine and human cells.