We have developed a technique for isolating nuclei and nuclear envelope(s) (NE) from Chinese hamster ovary (CHO) cells which does not depend on the use of detergents to solubilize contaminating cytoplasm. In our procedure NE are prepared from purified nuclei by nuclease digestion and subsequent high salt-sucrose gradient centrifugation. The nuclei and NE fractions are free of significant contamination by other subcellular organelles as judged by electron microscopy and enzyme analysis. Examination of the peptide and glycopeptide composition of the NE fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a very complex coomassie blue staining profile with prominent bands in the 55 000-75 000 molecular weight range. Using this NE isolation technique, we have examined the breakdown and re-formation of the NE during a limited stage (late G2, M, and early G1) of the replicative cycle in synchronized populations of CHO cells. Our data demonstrate that a minimum of 60% of the early G1 NE protein and a minimum of 50% of the early G1 NE phospholipid were present in the cell during the preceding G2 phase of the cell cycle and were reutilized in the re-formation of the NE occurring during late M and early G1. Our evidence suggests that the vast majority of the newly synthesized peptides and glycopeptides of the NE which appear in the daughter NE are synthesized during the early G1 phase of the replicative cycle. Examination of the NE peptides by one-dimensional gel electrophoresis suggests that no reproducible changes in NE peptide composition can be correlated with specific phases of the cell cycle.