Earlier studies (Soonpaa et al., 1996) revealed a rapid drop-off of ventricular cardiomyocyte cell-cycle activity at birth in mice, followed by a burst of DNA synthesis during the first week of postnatal life which contributed to the formation of multi-nucleated cardiomyocytes by postnatal day 10 (PN10). It has recently been suggested that a second burst of cardiomyocyte cell-cycle activity occurs during preadolescence, between PN14 and PN18, resulting in a 40% increase in cardiomyocyte number (Naqvi et al., 2014). Since there was no overt change in mono-versus bi-nuclear cardiomyocyte content and no change in cardiomyocyte nuclear ploidy between PN14 and PN18, a 40% increase in ventricular cardiomyocyte number during preadolescence should result in newly synthesized DNA in 57% of the cardiomyocyte nuclei. To characterize this putative burst of preadolescent cell-cycle activity, MHC-nLAC mice, expressing a nuclear-localized b-galactosidase reporter in cardiomyocytes and maintained in a DBA/2J background (Soonpaa et al., 1994), were implanted with BrdU-containing osmotic mini-pumps. Cumulative ventricular cardiomyocyte DNA synthesis was quantitated by co-localization of b-galactosidase and BrdU immune reactivity (Figure 1 A) as described (Reuter et al., 2014). Only low levels of cardiomyocyte DNA synthesis were detected in mice carrying pumps from PN10 through PN19 (2.96%±0.55%) or from PN12 through PN19 (1.09%±0.33%; see also Table S1A). BrdU was detected in small intestine crypt cells by 24 hr postimplantation, and at the end of the labeling period (Figure 1 B), confirming continuous infusion. Cumulative preadolescent cardiomyocyte DNA synthesis was also quantitated in C57Bl/6J inbred mice (the strain used by Naqvi and colleagues); S-phase cardiomyocytes were identified by nuclear BrdU immune reactivity in dispersed cell preparations (Figure 1 C). Only low rates of cardiomyocyte DNA synthesis were detected (Table S1B). Mice receiving a single BrdU injection on PN14. 5, PN15, or PN16 and analyzed on PN19 also had little labeling (Table S1C), arguing that BrdU cytotoxicity and/or the presence of the osmotic minipump per se were not confounding factors.