Human adenovirus type 12 (Ad12) replicates in permissive human host cells, but undergoes an abortive infection cycle in non-permissive hamster cells. Ad12 DNA cannot replicate and late viral genes are not expressed in hamster cells, whereas most of the early viral mRNAs are synthesized. We have shown previously that the major late promoter of Ad12 DNA (Ad12 MLP; nucleotides -228 to +435 relative to nucleotide +1 as the site of transcriptional initiation) does not function in uninfected or in Ad12-infected hamster BHK21 cells. The transcriptional defect of Ad12 DNA in hamster cells has thus been, at least partly, localized to the viral MLP. As expected, this construct is active in permissive human cells. Here, we show that the sequence between nucleotides +249 and +435 in the Ad12 MLP is in some way responsible for the late transcriptional block of this promoter in hamster cells. An Ad12 MLP--CAT construct comprising nucleotides -228 to +248 shows striking activity in hamster cells, and its activity is very markedly enhanced in Ad2- or Ad12-infected hamster or human cells compared with the nucleotide -228 to +435 construct. By using exonuclease Bal31, a series of Ad12 MLP--CAT gene assemblies were constructed which carry deletions of increasing lengths in the downstream part of the Ad12 MLP. Activity measurements of these constructs in BHK21 and in HeLa cells have located the presumptive mitigator element to the Ad12 sequence between nucleotides +320 and +352 of the MLP. It is also demonstrated that in the nucleotide -228 to +248 MLP construct, transcription is initiated at the authentic Ad12 MLP cap site after the transfection of both hamster and human cells. The localization of this cap site in the nucleotide sequence of the Ad12 MLP indicates the similarity to the comparable start site in the MLP of Ad2 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)