The E1 open reading frame of bovine papillomavirus type 1 (BPV) has been shown previously to encode trans-acting functions, M and R, that are involved in extrachromosomal replication of the viral genome. We have determined that several E1 mutants mapping in both the M and R regions and a single mutant of the upstream regulatory region have a higher transforming activity on mouse C127 cells than the wild-type genome does. A representative mutant in M, a mutant in R, and the upstream regulatory region mutant were complemented in trans by the wild-type genome, but the two E1 mutants did not complement each other, suggesting that they affect the same inhibitory function. A long terminal repeat-activated clone constructed to express the intact E1 open reading frame reversed the high-transformation phenotype of the mutants. In contrast to the high-copy-number autonomous replication of the wild-type genome, the genomes of the E1 mutants were, as previously described for other E1 mutants, integrated at lower copy numbers in the transformed cells. Relative to the viral genome copy number, both the E1 M and R mutant transformed cells contained an average of 10-fold more BPV-specific transcripts than did the wild-type transformed cells. Cycloheximide treatment of the cells transformed by the E1 mutants did not lead to the rapid 10-fold increase in the accumulation of viral transcripts observed with the wild-type genome. These results suggest either that integration of the BPV genome makes it unresponsive to a labile repressor or that an E1 gene product, containing both M and R sequences, is a repressor of BPV transcription.