Simian virus 40 nucleoprotein complexes (SV40 chromatin) have a nucleosome structure similar to that of cellular chromatin and serve as a useful model system for studying structure and function of cellular chromatin. It is the purpose of this study to clarify the molecular forms of SV40 chromatin and their functional changes. African green monkey kidney cells (CV-1) were infected with SV40 and bulk or pulse-labeled with [(3)H] thymidine. SV40 chromatin was isolated under various conditions from these cell nuclei and examined by electron microscopy. When SV40 chromatin was extracted with a hypertonic salt-Triton X-100 solution from the nuclei isolated by treatment with a hypotonic ethylenediaminetetraacetic acid (EDTA) solution, the majority of SV40 chromatin molecules had a relaxed form showing a typical bead-like structure of nucleosomes. Most of these molecules were found to be derived from disrupted virions. When the extraction was performed under mild conditions in a near isotonic solution by no pretreatment with EDTA, SV40 chromatin and virions were separated in different fractions by sucrose density gradient centrifugation. The majority of these SV40 chromatin molecules had a condensed form. These molecules can be relaxed through various intermediate forms by treatment with a hypotonic or 0.6M NaCl solution. Replicating SV40 chromatin was sedimented faster than mature SV40 chromatin and showed the relaxed form. SV40 chromatin isolated from purified virion by alkali treatment showed the relaxed form. The relaxed molecules were condensed and their template activity for transcription was strongly inhibited by binding with histone Hl.