This study was designed to examine effects of theophylline, a methylxanthine, on both the positive inotropic and toxic actions of cardiotonic steroids in cardiac muscle isolated from guinea pig heart. In electrically paced left atrial muscle, 0.3 mM theophylline reduced both the maximum developed tension observed in the presence of increasing concentrations of strophanthidin and the dose of this steroid that first elicited extrasystoles. Similarly, 0.3 mM theophylline decreased the time to onset of arrhythmias produced by 5 microM digoxin and the fractional occupancy of specific binding sites on Na,K-adenosine triphosphatase by digoxin at the onset of these dysrhythmic events. A higher level of theophylline (6.5 mM) severely diminished or prevented the positive inotropic and arrhythmogenic actions of cardiotonic steroids while promoting the contracture elicited by these digitalis-like compounds. In spite of the severe contracture observed in the presence of 6.5 mM theophylline plus 5 microM digoxin, the digoxin fractional occupancy was significantly less than that observed at the onset of digoxin-induced extrasystoles and contracture in the absence of theophylline. In radiolabeled ligand binding experiments, 6.5 mM theophylline reduced the affinity of specific binding sites for ouabain while having no effect on receptor density. These results, when considered in light of previous reports by other investigators, suggest that moderate concentrations of methylxanthines promote cardiotonic steroid-induced arrhythmias by increasing Ca++ influx and its uptake into sarcoplasmic reticulum. Higher levels seem to antagonize the arrhythmogenic actions by inhibition of sarcoplasmic reticular Ca++ uptake and by antagonism of receptor binding.