Background: Compensatory renal growth consists of cellular enlargement and a small but consistent increase in DNA content. It has been assumed that the increase in the total renal DNA content was due to new cell formation. Methods: To test the hypothesis of whether cellular hyperplasia is the cause of the compensatory renal growth after the loss of the renal parenchyma and the timing of the DNA increase in neonatal rats, we performed cell cycle analysis using flow cytometry. Results: Following unilateral nephrectomy, the maximum increases of neonatal cortical cells entering the S phase occurred at 72 and 120 h (9.4 and 9.6% compared to 7.0 and 6.1% of the sham-operated group). Peak increases of neonatal kidney cortical cells entering the G2M phase occurred at 48 and 72 h (4.3 and 4.6% compared to 3.3 and 3.9% of the sham-operated group). Conclusion: DNA synthesis and replication occurs during compensatory renal growth following unilateral nephrectomy in neonatal rats as evidenced by an increase in cells entering both the S and G2M phases. In neonatal rats these events appear to be completed within 48–120 h after nephrectomy.