Wild type polyomavirus expression is suppressed in embryonal carcinoma (EC) cell lines. This suppression is alleviated when the EC cells are induced to differentiate. Several characterized host range mutants of polyoma overcome suppression and are able to express and replicate in the undifferentiated EC cells. These previously described isolates were obtained by serial passage of a wild type strain through PCC4 or F9 EC lines. We present a new pyPCC4 isolate (LPT) derived without selection in EC cells. Isolates with host range specificity for a given EC line have been reported to share several common rearrangements and features. These features are also observed in LPT. We report a novel feature shared by these mutants, including LPT, capable of expression in the EC cell line PCC4. In 8 of 10 isolates a novel sequence is created within the enhancer region by rearrangement junctions with near perfect homology to the AP-1 core consensus sequence, 'TGACT(C/A)A'. That the precise location of these junctions varies among these isolates suggest a functional role for this conserved sequence. Our goal is to understand the function of various mutations in host range mutants of polyoma. In order to understand the rearrangements necessary for expression and replication of polyoma in PCC4 cells, we have further characterized the limits of the B enhancer in these cells as compared to those described in permissive cell systems. We have been able to locate the origin proximal limit of the B enhancer for replication close to nt 5189 and distinguish it from the origin proximal limit of the B enhancer for transcription near nt 5215. The two B enhancer cores overlap but do not coincide and are conserved in both cell lines.