Simian virus 40 (SV40) chromatin and mature virion were extracted from formaldehyde-fixed SV40-infected CV-1 cells. Mature virion was eliminated from SV40 chromatin by sucrose density gradient centrifugation. Replicating and nonreplicating SV40 chromatin were separated on CsCl/ guanidine-HCl density gradients. Replicating SV40 chromatin was lower in density than the nonreplicating form. The analysis of chromatin proteins by polyacrylamide gel electrophoresis and silver staining revealed that replicating chromatin has less H1 histone than the nonreplicating form.