During exercise there is an increase in the circulating levels of catecholamines and enhanced sympathetic tone (Scheurink et al. 1989). As a result, several studies have documented beta-adrenergic receptor (β-AR) modification which may represent one of the mechanisms underlying the relative bradycardia in trained subjects (Werle et al. 1990). In light of this, we investigated whether the response of β-AR to stimulation was altered in a model of voluntary wheel running exercise in female rats (Natali et al. 2002). Following 6-7 weeks of voluntary running, heart weight: body weight was significantly greater in trained (TRN) than sedentary (SED) animals (5.6 ± 0.1 vs 4.8 ± 0.1, TRN vs SED, respectively, (mean ± SEM, P<0.001, unpaired t test, n = 24 in each group). Left ventricular myocytes were isolated from the hearts of TRN and SED rats and field stimulated to evoke contraction at a frequency of 1 Hz at 37°C. Contraction magnitude was measured optically and was expressed as a percentage of resting cell length. Selective β1-AR stimulation (isoprenaline, 10^-7 M, in the presence of the β2-AR antagonist, ICI 118,551, 10^-7 M) was unaltered by exercise but the inotropic response to β2-AR stimulation (salbutamol, 5×10^-5 M, in the presence of β1-AR blockade, atenolol 10^-7 M) was significantly depressed in TRN cells (50.0 ± 6.8 vs 18.5 ± 8.0%, SED vs TRN, respectively, P < 0.05, unpaired t test, n = 47-54 cells). β2-AR stimulation activates both G_i and G_s signalling cascades. To investigate whether the reduced response to β2-AR stimulation in the TRN myocytes was a result of enhanced G_i signalling, left-ventricular myocytes were incubated in pertussis toxin (PTX, 2-3 hrs at 37°C) to inhibit G_i protein function. The contractile response to β2-AR stimulation was enhanced in both TRN and SED groups following PTX pre-treatment to a similar level, i.e. the depressive effect of exercise on the inotropic response to β2-AR was abolished by PTX (72.6 ± 10.5 vs 68.6 ± 9.8%, SED vs. TRN, respectively, NS, n= 31 – 32 cells). We conclude that voluntary exercise training induces a decrease in the positive inotropic response to β2-AR stimulation and this is at least in part due to enhanced G_i signalling. These effects contrast with the depression in β1-AR responsiveness with maintained β2 responsiveness reported in failing myocardium.