In the heart, Ca²⁺ release from the sarcoplasmic reticulum (SR) may play an important role in pacemaking in the sino-atrial node (SAN): it has been suggested that a rise of sub-sarcolemmal [Ca²⁺] due to spontaneous Ca²⁺ release from the SR activates inward Na⁺/Ca²⁺ exchange current, thus triggering the action potential (Hüser et al, 2000; Bogdanov et al, 2001). However, comparatively little is known about intracellular Ca²⁺ in the atrio-ventricular node (AVN; Hancox et al, 1994), which also exhibits pacemaker activity. We have therefore investigated the spatio-temporal characteristics of intracellular Ca²⁺ in myocytes isolated from the AVN. Myocytes were loaded with the Ca²⁺-sensitive fluorescent indicator fluo-3 AM (10 μM for 30 min) and imaged at room temperature using a confocal laser scanning microscope. The myocytes showed spontaneous contractions (0.5 ± 0.1 Hz; mean ± sem; n = 13). Transverse and longitudinal line scan images obtained from 1 μm sections through the centre of the cells revealed that the systolic Ca²⁺ transient was spatially inhomogeneous, with some areas showing little rise of intracellular Ca²⁺. The regions that showed an increase, or no increase, of [Ca²⁺] were consistent from beat-to-beat. When plotted against time, mean fluorescence for each line scan revealed a rapid rise of intracellular Ca²⁺ during the systolic Ca²⁺ transient (time to peak: 86.8 ± 7.8 ms; n = 13) followed by a slower decline (time constant: 182.7 ± 11.0 ms; n = 13); the transient was frequently (77.9 ± 7.1 %) preceded by a slow ramp increase of [Ca²⁺] lasting 234.4 ± 32.4 ms (n = 13). Immunohistochemistry showed the ryanodine receptors (RyR) located in regular transverse bands, as reported previously in other cardiac cell types. These data suggest that Ca²⁺ handling in AVN cells may be similar to that reported previously in SAN cells; the spatial inhomogeneity does not appear to be due to inhomogeneous RyR distribution but may be due to local differences in Ca²⁺ entry or RyR sensitivity.