In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.