The pulmonary vein is widely regarded as an important source of ectopic activity triggering atrial arrhythmias (Haïssaguerre et al., 1998). In many mammalian species, the vein is surrounded by a sleeve of cardiac-like cells that extends from the atria to the lungs (Best et al., 1961). These cardiac-like cells show intrinsic electrical activity (Chen et al., 2000); however, the mechanisms underlying the initiation of this activity remain unclear. It has been suggested that intracellular Ca²⁺ may influence the electrical activity in cardiac-like cells, thus we have examined Ca²⁺ signalling events in the pulmonary vein. Male Sprague Dawley rats (180-300g) were sacrificed by cervical dislocation. The pulmonary vein (0.7-1 cm long and 0.3-1 mm outside diameter) was dissected and loaded with 10 µM fluo-4 AM. The tissue was then placed in bath solution comprising (in mM); NaCl, 150; KCl, 5.4; HEPES, 10; Glucose, 10; MgCl₂, 1.2; CaCl₂, 1.8, pH 7.4 with NaOH, and imaged using a Zeiss Axioskop microscope equipped with a Hamamatsu CCD camera. Images were acquired using WinFluor (University of Strathclyde) and fluorescence was expressed as ΔF/F_min, with n being the number of tissues studied. Student’s paired t-test was used for statistical comparison and P<0.05 was considered to be significant. In all control tissues, spontaneous Ca²⁺ transients were observed within individual cardiac-like cells. The activity was generally asynchronous in nature yielding a mean frequency of 1.4 ± 0.02 Hz and amplitude ΔF/F_min=0.11 ± 0.01 (n=47). Upon removal of extracellular Ca²⁺ spontaneous activity continued, although there was a small but significant reduction in both the frequency (from 1.4 ± 0.1 to 1.1 ± 0.1 Hz, n=7, P<0.05) and amplitude (from ΔF/F_min=0.11 ± 0.02 to 0.09 ± 0.02, P<0.05). However, when the SR was depleted of Ca²⁺ by continued exposure to caffeine (20 mM), the spontaneous Ca²⁺ transients were almost completely abolished, with the frequency being reduced from 1.4 ± 0.1 to 0.03 ± 0.03 Hz (n=7, P<0.01). In the presence of ryanodine (20 μM) the frequency was reduced from 1.4 ± 0.1 to 0.1 ± 0.04 Hz (n=7, P<0.01), and the amplitude from ΔF/F_min=0.15 ± 0.03 to 0.02 ± 0.01 (P<0.01). The IP₃ receptor blocker 2-aminoethoxydiphenyl borate (20 µM) also reduced the frequency from 1.6 ± 0.2 to 0.4 ± 0.2 Hz (n=5, P<0.05) and amplitude from ΔF/F_min=0.05 ± 0.01 to 0.01 ± 0.004 (P<0.05). Inhibiting the SR Ca²⁺ ATPase with cyclopiazonic acid (20 µM) produced a decease in frequency from 1.4 ± 0.2 to 0.2 ± 0.1 Hz (n=7, P<0.01) and amplitude from 0.10 ± 0.03 to 0.03 ± 0.02 ΔF/F_min (P<0.05). This study has shown that spontaneous Ca²⁺ transients occur in individual cardiac-like cells of the pulmonary vein under resting conditions. Furthermore, our results indicate that SR Ca²⁺ release via both the ryanodine and IP₃ receptors is important for producing this spontaneous activity.