A fresh inoculum of human adenovirus type 12 (Ad12) was obtained from the American Type Culture Collection and passaged once on human embryonic kidney cells, and Ad12 DNA was prepared from the first-passage yield to avoid higher passages which might have generated host-virus DNA recombinants. The 18 PstI fragments of Ad12 DNA were cloned into the pBluescript KS vector, and the entire nucleotide sequence of both strands from all 18 fragments was determined by using successive oligodeoxyribonucleotide primers. Ad12 DNA extends over 34,125 nucleotide pairs, and its molecular weight is calculated to be about 22 x 10(6). The nucleotide sequence of Ad12 DNA was subjected to computer analyses that determined possible open reading of frames on the two strands, the leader sequences, the position of the virus-associated RNA coding region, possible TATA, and polyadenylation signals. The distribution of the Ad12 open reading frames was similar to that in the previously sequenced Ad2 DNA, but there were also distinct differences. Ad12 DNA has an inverted terminal redundancy of 161 nucleotides, compared with 102 nucleotides in Ad2 DNA. There were stretches of sequence identity between Ad2 and Ad12 DNAs at both termini; the overall sequence similarity between the two viral genomes ranged between 59% (polypeptide IX) and 77% (in the E2 region), with high homology also in the sequences for the adenovirus DNA polymerase.