Human adenovirus mutants that carry a large deletion in early region 4 (E4) are severely defective in the synthesis of viral late proteins. Plasmids that carry intact E4 sequences can complement the late protein synthetic defect of such mutants when introduced into infected cells by transfection, presumably due to the transient expression of E4 products. Cells transfected with cDNA clones capable of expressing E4 open reading frame (ORF) 6, or deletion mutant clones expected to express either E4 ORF 6 or E4 ORF 3, also complement the mutants' defects. Thus, these E4 ORFs can individually satisfy the requirement for E4 products in viral late gene expression, and function effectively in the absence of other E4 products. Some E4 deletion mutants also exhibit a defect in the production of viral DNA. All of the clones that stimulate late gene expression also enhance one such mutant's ability to accumulate viral DNA. Thus, the ORF 3 and ORF 6 products are also individually sufficient to provide an E4 function necessary for normal viral DNA replication in the absence of other E4 products.