1. Decreasing heart rate during development is known to be the result of parasympathetic nervous system maturation that depresses the pacemaker current (If) by acetylcholine (ACh). However, a direct effect of ACh on If has been ruled out and the involvement of other secondary messengers, such as cAMP, was verified in previous studies. Therefore, we hypothesized that reduced basal cAMP production in sinoatrial (SA) nodal cells may contribute to the slowing of heart rate after birth. 2. The electrocardiogram and heart rate variability (HRV) were documented and measured in vivo and in vitro (in isolated perfused Langendorff preparations) for rabbits aged 2, 4, 6, 8 and 12 weeks. Sinoatrial node action potential (AP) recording and perforated patch-clamp analyses were used to investigate the spontaneous depolarization rate and pacemaker If currents. Concentrations of cAMP in SA nodal tissues were determined by radioimmunoassay. Relative expression of adenylate cyclases (ADCY1, 5) and phosphodiesterases (PDE1A, 4A and 8A) were quantified by real-time reverse transcription-polymerase chain reaction. 3. Significantly reduced heart rate, but unchanged HRV, was observed in perfused hearts in the older age groups, accompanied with a slowed phase 4 spontaneous depolarization rate (90.5 +/- 4.7 vs 49.6 +/- 2.6 mV/s for 2 week vs 4 week hearts, respectively; n = 5; P < 0.05), a negative shift of the If threshold potential (-45.5 +/- 3.0 vs -51.1 +/- 6.0 mV for 2 week vs 4 week hearts, respectively; n = 9; P < 0.05) and decreasing basal levels of SA nodal cAMP (0.31 +/- 0.05 vs 0.025 +/- 0.002 micromol/L for 2 week vs 4 week hearts, respectively; n = 6; P < 0.05). Gene expression levels of PDE1A, 4A and 8A were increased in the 12 week group compared with the 2 week group 1.5-, 2- and 1.8-fold, respectively (P < 0.05), with little change in ADCY1 and 5. 4. These data suggest that, in addition to autonomic innervation, slowing of heart rate during postnatal maturation can be attributed to a negative shift of the If activation caused by diminished baseline cAMP content in SA nodal cells.