The Na⁺–Ca²⁺ exchanger in smooth muscle from the pulmonary artery, is known to be involved in regulating [Ca²⁺]_i (Wang et al. 2000). However, the role of the Na⁺–Ca²⁺ exchanger in regulating [Ca²⁺]_i in the pulmonary vein (PV) has received little attention. In the PV, two phenotypically distinct myocytes have been reported (see Michelakis et al. 2001). Thus, we have examined how [Ca²⁺]_i signalling in these two distinct myocyte phenotypes is affected by inhibition of the Na⁺–Ca²⁺ exchanger. Male Sprague-Dawley rats (200-300g) were humanely killed. Myocytes were isolated from intrapulmonary veins (< 400 μm), using a procedure similar to that described by Drummond & Tuft (1999) for the pulmonary artery, and thereafter incubated with 5 μM fura-2 AM. To achieve a Na⁺-free extracellular solution, equimolar LiCl replaced NaCl. All experiments were carried out at room temperature. Mean data±S.E.M. are given and n is the number of cells. Statistical differences were tested for using Student’s paired t test. P < 0.05 was considered to be significant. In cardiac-like myocytes isolated from the vein, spontaneous oscillations in [Ca²⁺]_i were observed, with a frequency of 0.3 ±0.1 Hz. Replacing Na⁺ in the bath solution with Li⁺ resulted in an increase in basal [Ca²⁺]_i from 72±17 nM to 156 ±46 nM (P< 0.05, n=6), and the frequency of spontaneous oscillations also increased to 0.6±0.1 Hz (P< 0.05, n=6). A small but significant decrease in peak [Ca²⁺]_i during oscillations was observed in Na⁺-free bath solution (132±3 nM to 124±4 nM, P< 0.05, n=6). Caffeine-induced [Ca²⁺]_i transients were significantly increased from 755±133 nM to 1150±230 nM (n=6, P<0.05) in Na⁺-free bath solution, and the time for 90% recovery (t_{90% recovery}) was increased from 56±8s to 132±16s (P<0.05). Spontaneous oscillations in [Ca²⁺]_i were absent in typical spindle shaped smooth muscle cells isolated from the vein. Application of caffeine (20 mM) or ATP (10 μM) transiently increased [Ca²⁺]_i to 759±24 nM and 556±32 nM, respectively. In Na⁺-free solution, there was no change in basal Ca²⁺. However, both caffeine and ATP-induced [Ca²⁺]_i transients were increased (13 ±3% and 36±5%, respectively). The t_{90% recovery} also increased from 9±2s to 15±5s for caffeine and from 12±3s to 26±4s for ATP (P< 0.05). In 60% of cells, the frequency of ATP (10 μM) induced [Ca²⁺]_i oscillations was increased in Na⁺-free bath solution. These results suggest an important role for the Na⁺–Ca²⁺ exchanger in regulating [Ca²⁺]_i signalling in the two distinct phenotypes of myocytes found in the pulmonary vein.