The structure of minichromosomes from an SV40 recombinant carrying a functional mouse alpha-globulin gene in its late coding region was studied by DNase I treatment of isolated nuclei from infected cells. Ten prominent DNase-I-hypersensitive sites were detected in the inserted globin fragment, six in the region upstream from the transcription initiation site and four within the gene itself. Corresponding sites were not seen in naked DNA, nor in minichromosomes without this insertion. However this hypersensitivity pattern differs from that observed for mouse alpha-globin in Friend erythroleukemia cells before or after induction. The recombinant minichromosome also displayed eight hypersensitive sites, indistinguishable from those seen in unsubstituted SV40, in the viral sequences containing the origin of replication and the regulatory signals for early and late viral transcription. The cleavage sites in SV40 occur predominently in the origin-promotor-enhancer region, whereas those in the globin fragment were more dispersed and less pronounced. This suggests that the specific local opening of this region in the viral chromatin is not due solely to the presence of active promoters, but is associated with the function of the enhancer element.