The nucleoside triphosphate diphosphohydrolases (NTPDase; EC 3.6.1. 5) are a family of ectonucleotidases associated with vascular endothelial and smooth muscle cells. These ectonucleotidases are involved in the control of vascular tone by regulating the level of circulating ATP. Ca(2+)-channel blocking agents are currently used for the treatment of hypertension. Considering the external localization of the NTPDase catalytic site and its Ca(2+) requirement for enzyme activity, a possible interference of calcium antagonists (nifedipine, verapamil-HCl, and diltiazem-HCl and some of its metabolites) could be anticipated. To test that hypothesis, an NTPDase-enriched particulate fraction was used. Our results show that verapamil, diltiazem, and its metabolites all produced a concentration-dependent inhibition of NTPDase, at concentrations greater or equal to 0.1 mM with verapamil and to 0.5 mM with diltiazem and its metabolites, whereas no significant effect was observed with nifedipine. Kinetic studies, carried out to define the mode of action of these drugs, showed a mixed type of inhibition. Based on their respective K(i) values (in parentheses, in mM), inhibitory potencies of these molecules were in the following order: desacetyl-N-desmethyldiltiazem (M(2)-HCl; 0.6) > verapamil (0.76) > N-desmethyldiltiazem (M(A;) 0.9) > diltiazem (2.4) > desacetyl-O-desmethyldiltiazem (M(4)-HCl; 3.5) > desacetyl N, O-desmethyldiltiazem (M(6)-HCl; 3.9). Hence, these calcium antagonists can be considered as weak NTPDase inhibitors. Moreover, based on these K(i) values and the range of concentrations found in the blood, NTPDase would not be inhibited significantly in vivo.