The inotropic actions of xanthine derivatives with long alkyl chains were investigated in guinea pig ventricular papillary muscle. A potent and nonselective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine, elicited a positive inotropy and inhibited the negative inotropic effects of calcium channel inhibitors, as did a selective PDE III inhibitor, amrinone, and these effects were canceled by a protein kinase inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). However, 1,3-di-n-butyl-7-(2'oxopropyl)xanthine (denbufylline) and 1-n-butyl-3-n-propylxanthine (XT-044), which have potent and selective PDE IV-inhibitory activities, showed negative inotropic actions that became more potent in the presence of H-89. Denbufylline abolished the late restoration phase induced by ryanodine. This xanthine derivative attenuated the effects of both the calcium channel acting agents Bay K 8644 and verapamil, without interaction with caffeine and dihydropyridine calcium channel inhibitors, and denbufylline had little direct influence on the specific binding of [(3)H]azidopine and [(3)H]desmethoxyverapamil to cardiac membranes. A nonxanthine PDE IV inhibitor, Ro 20-1724, did not affect the inotropic actions of calcium channel inhibitors. The attenuation by denbufylline or XT-044 of the negative inotropic action of verapamil was not influenced by treatment with H-89. These results suggest that in the ventricular papillary muscle, these xanthine derivatives elicit negative inotropy by acting on a verapamil-sensitive site of the calcium channel without involving their PDE-inhibitory activity.