A number of Cl^– currents have been identified in cardiac myocytes and these may contribute both depolarising inward current and repolarising outward current. Of these, a current activated by the cAMP/protein kinase A (PKA) pathway has been suggested to play a role in action potential repolarisation in the presence of β-adrenoceptor agonism. A cardiac isoform of the cystic fibrosis transmembrane conductance regulator, cftr, has been suggested to contribute to this current. Considerable heterogeneity in the functional expression of certain ion channels has been shown between the t-tubule membrane and the surface sarcolemma. However, there is very little information regarding the localization of PKA-dependent Cl^– channels in cardiac myocytes. We have attempted to address this question using a combination of scanning ion conductance microscopy and cell-attached recording (Gu et al., 2005), from isolated guinea-pig ventricular myocytes. Guinea-pig cardiomyocytes were isolated by mechanical disruption following retrograde perfusion of the aorta with a collagenase-containing Tyrode’s solution. Pipettes (typically 15-30 MΩ) were filled with a recording solution in which permeant cations had been replaced with N-methyl-D-glutamate and cells superfused with a normal Tyrode’s solution at room temperature. Using the pipette current as a feedback signal, 10μmx10μm scans of the cell surface were conducted. Data are presented as mean ± SEM. Grooves in the cell surface with a spacing of 2.0±0.1μm (n=30) could be identified, which were assumed to correspond to the z-line grooves. The pipette was then directed to a location on the cell surface identified in the image and, after formation of the giga-seal, on-cell recordings were made. Cells were superfused with an activating cocktail of 5μM isoprenaline/10μM forskolin/50μM isobutyl methylxanthine (IBMX), which activated anion-selective currents in 6/38 recordings. Membrane potentials within the patch were calculated as the difference between the resting membrane potential (RMP) and the pipette potential, assuming RMP = -80mV. Currents were typically large and noisy, making it difficult to distinguish unitary channel currents. The mean pipette current ranged from 90.8±14.3pA at +100mV to -27.1±3.6pA at -80mV and reversed at -32.3±2.6mV (n=6). Current activity was lost on excision of the patch and, in 2 patches where this was attempted, could be recovered by application of PKA catalytic subunit to the cytosolic surface. While the majority of current were recorded from the ‘scallop’ between z-grooves, current activity was also recorded within the z-grooves. These data are consistent with the suggestion that PKA-activated Cl^– channels occur in clusters containing >100 channels within the sarcolemma of guinea-pig ventricular myocytes.