Ageing is a major risk factor for the development of heart failure and arrhythmias. The inward rectifier potassium current, I_K1, is crucial for terminal phase repolarisation and stabilising the resting membrane potential of cardiac myocytes. Reductions in I_K1 contribute to an increased susceptibility to arrhythmias in heart failure (Pogwizd et al. 2001). Previously, we have shown an increase of I_K1 in left ventricular midmyocardial myocytes that may reduce susceptibility to arrythmias. We investigated the expression of I_K1 across the left ventricular free wall of young and old sheep as well as the sensitivity of I_K1 to β-adrenergic stimulation. Young adult (18 months old) and old sheep (>8 years) were euthanased with 200 mg.kg^-1 intravenous pentobarbitone. Myocytes were isolated from the endo-, midmyo- and epicardial layers of the left ventricular free wall by collagenase digestion. I_K1 currents were recorded using the whole-cell voltage clamp technique with a K⁺-based pipette solution at 37^oC. Statistical analyses were carried out using 1-way ANOVA or a Mann-Whitney rank sum test for non-normally distributed populations. Steady-state I_K1 was uniform across the wall in young hearts, whereas the old hearts had developed a transmural I_K1 gradient (endo > mid ≈ epi; P < 0.05). The I_K1 gradient in the old hearts was due to a greater channel conductance and a rightward shift in Vmid in the endocardial myocytes (P < 0.05), with no difference in slope conductance (Vc). Steady-state I_K1 in both endo- and midmyocardial myocytes was increased in the old sheep (young vs old, mid: -1.31 ± 0.10 vs -2.00 ± 0.18 pA/pF, n = 20 and 27; P < 0.05; endo: -1.55 ± 0.16 vs -5.09 ± 1.13 pA/pF, n = 10 and 11; P < 0.05). These changes were accompanied by an increased channel conductance and a rightward shift in Vmid, with a decrease in slope conductance (Vc). β-adrenergic stimulation using isoprenaline (100nM) reduced steady-state I_K1 in old endocardial myocytes only (-5.09 ± 1.13 vs -2.22 ± 0.30 pA/pF, n = 11 and 8; P < 0.05). Channel conductance was decreased with isoprenaline with no change in Vmid, but an increase in slope conductance (Vc). β-adrenergic stimulation reduced steady-state I_K1 to amplitudes similar to those in old epi- and midmyocardial myocytes and was sufficient to eliminate the steady-state I_K1 transmural gradient present in the old hearts. In conclusion, we show that I_K1 is remodelled in aged ventricular myocytes. Increases in I_K1 in the endo- and midmyocardial layers may be an important mechanism in stabilising the resting membrane potential and limiting the prolongation of the action potential duration associated with ageing (Dibb et al. 2004). β-adrenergic-induced inhibition of I_K1 may increase the arrythmogenic potential in endocardial myocytes of old sheep by preventing this mechanism thus increasing the dispersion of repolarisation in the old heart.