Protein accumulation was measured in human NHIK 3025 cells, synchronized by mitotic selection, during and following treatment with extreme hypoxia (less than 4 ppm O2). Comparison was made between cells treated while in different stages of the G1 phase. The total protein content was measured in individual cells and related to cell cycle phase by coincident recording of DNA and protein using two-parametric flow cytometry. After a 24 h treatment with extreme hypoxia, all cells, irrespective of where in G1 they were located at the start of treatment, were reversibly arrested in a pre NDA synthetic stage. During this treatment there was a slight reduction (approximately 2%) in the average amount of protein per cell. The arrested cells were found to display a heterogeneous protein content after treatment, but cells rendered hypoxic while in early-G1, had clearly lost more protein during hypoxia than cells rendered hypoxic while in late-G1. After reoxygeneration, the cells initiated DNA synthesis after first having restored the amount of protein which is normal for these cells at the stage of initiation. Thus, in the first hours following reoxygenation, cells rendered hypoxic while in early-G1 accumulated protein faster than those rendered hypoxic while in late-G1 (the respective rates being 12.3 versus 5.2%/h). We conclude that this protein accumulation may be of importance in restoring the pool of protein required for the initiation of DNA synthesis and that the differences in rate reflect the amount of protein lost during hypoxia.