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A direct quantitative method was developed for detecting a small fraction of minichromosomes active in transcription and for analyzing their chromatin structure. For this, SV40 minichromosomes were incubated in vitro in the presence of [alpha‐32P]UTP to allow elongation of the in vivo pre‐initiated nascent RNA and thus labeling of the in vivo transcriptionally active molecules. Subsequently the minichromosomes were treated with Sarkosyl to remove most of the proteins bound to the viral DNA while leaving the 32P‐labeled RNA‐DNA‐polymerase ternary complexes intact. Following agarose gel electrophoresis, the bulk SV40 DNA was identified by staining with ethidium bromide and the labeled ternary complexes by autoradiography, although they are not well separated in the gel. When the viral minichromosomes were cleaved with certain restriction enzymes or mildly with DNase I prior to the in vitro labeling and Sarkosyl treatment, analysis by gel electrophoresis showed that all transcriptionally active minichromosomes were cleaved, whereas a large fraction of the bulk minichromosomes was resistant to cleavage. In all molecules the cleavage occurred within the transcriptional regulatory region. In addition, analysis by electron microscopy revealed that SV40 minichromosomes having a nucleosome‐free region (gap) are also more sensitive to the initial cleavage by both DNase I and Bg/I restriction enzyme than non‐gapped molecules and that the initial DNase I cleavage occurs within the gap. Thus, at least in SV40, the nuclease‐hypersensitive region is positively correlated with the existence of a gap.

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