The full-length product of the bovine papillomavirus type 1 (BPV-1) E1 translational open reading frame is required for viral DNA replication in vivo and in vitro. E1 is a multifunctional protein whose properties include ATP binding, acting as an ATPase-dependent DNA helicase, DNA binding to the BPV-1 origin of viral DNA replication, and association with the E2 transcriptional transactivator, E2TA, a second viral protein involved in DNA replication. All of these properties are thought to be important for E1's role in replicating the viral genome. In addition BPV-1 E1 can inhibit activation of the viral P89 promoter by the BPV-1 E2TA. E1 has amino acid homology with eight regions of SV40 large tumor antigen (T-ag), a DNA helicase that is essential for the replication of the SV40 DNA genome. These eight regions of similarity lie within the domain of T-ag that confers DNA helicase activity. We created a series of missense mutations in BPV-1 E1 at codons 295, 344-345, 446, 464, 466, 497-498, 523, and 542, which encode amino acids of identity in seven of the eight regions of similarity between E1 and T-ag, and at codon 370. The activities of these mutant E1 genes were compared to wild-type E1 in multiple assays that measured DNA replication, inhibition of E2TA-dependent transcription, DNA binding, ATP binding, and protein expression. Based upon these analyses, the following conclusions were made: (i) at least five of the eight regions in E1 that are similar to regions in T-ag are functionally important in viral DNA replication; (ii) specific E1 missense mutants, themselves defective for supporting DNA replication, could act in trans to suppress the replication function of wild-type E1; (iii) certain regions of similarity with T-ag that are important for E1's ability to support DNA replication are not necessary for its capacity to inhibit E2TA-dependent transcription; and (iv) efficient DNA binding by E1 is not essential for E1 to inhibit E2TA-dependent transcription.