L-type Ca2+ current (I(Ca,L)) facilitation is a dynamic pattern of increased peak current and slowed inactivation evoked by repetitive stimulation after rest or increasing frequency of stimulation. Facilitation of I(Ca,L) is an important mechanism for afterdepolarizations in ventricular myocytes and may also contribute to post-rest potentiation. I(Ca,L) also contributes to the generation and modulation of the pacemaker action potential in sinoatrial nodal (SAN) cells. However, the presence and the potential role of I(Ca,L) facilitation in SAN cells is less clear. We measured I(Ca,L) in regularly-beating SAN cells isolated enzymatically from wild-type mice using the whole-cell patch-clamp technique. I(Ca,L) was confirmed by its sensitivity to Nifedipine 5 μM and Isoproterenol 100 nM. The train of the test depolarizations evoked at 0 or 10 mV from a holding potential of -80 mV was applied at 0.5 Hz after at least 3 minutes rest at -80 mV. I(Ca,L) was recorded with Na+ and K+ free (TEA+ substitute for Na+) bath solution and 10 mM EGTA with 3 or 0 mM Ca2+ in the pipette solution. There were no significant changes in SAN cell I(Ca,L) amplitude or inactivation kinetics during a train of repetitive stimuli with 10 mM EGTA and 0 mM Ca2+ in the pipette solution (estimated [Ca2+]i 0.15 pM assuming 10 nM Ca2+ contamination in Millipore water). The average maximal increase in the time integral of I(Ca,L) was 5±2%, and 2±3% in the peak amplitude of I(Ca,L) (n=10). In contrast, I(Ca,L) was dramatically facilitated in ventricular myocytes tested under the same experimental conditions (74±16% increase in the time integral of I(Ca,L) and 18±3% increase in the peak amplitude of I(Ca,L) (n=7)). However, I(Ca,L) facilitation (21±8% increase in integrated I(Ca,L) and 18±5% increase in the peak amplitude of I(Ca,L), n=12) was observed in SAN cells when 3 mM Ca2+ was added to the pipette solution (estimated [Ca2+]i 62 nM). I(Ca,L) facilitation in ventricular myocytes was 94±7% increase in integrated I(Ca,L) and 26±2% increase in the peak I(Ca,L) ( n=9 ) with 3 mM Ca2+ under the same experimental conditions. Facilitation in SAN cells was abolished by pretreatment with ryanodine 10 μM (-3±4% in integrated I(Ca,L) and 1±2% in peak amplitude of I(Ca,L), n=5 ). Facilitation is also abolished by ryanodine in ventricular myocytes (Wu et al. (2001)). These results show that I(Ca,L) facilitation in mouse SAN cells is less robust and occurs in a narrower window of [Ca2+]i compared to ventricular myocytes. These findings suggest that the dominant L-type calcium channel (CaV1.3) in SAN cells might have a different mechanism of facilitation compared to ventricular myocytes.