The cytoplasmic domain linking transmembrane segments 2 and 3 (LOOP1) of the yeast H(+)-ATPase was probed by the introduction of unique factor Xa recognition sites. Three sites, I170EGR, I254EGR and I275EGR, representing different structural regions of the LOOP1 domain, were engineered by site-specific mutagenesis of the PMA1 gene. In each case, multiple amino acid substitutions were required to form the factor Xa sites, which enabled an analysis of clustered mutations. Both I170EGR and I275EGR-containing mutants grew at normal rates, but showed prominent growth resistance to hygromycin B and sensitivity to low external pH. The engineered I254EGR site within the predicted beta-strand region produced a recessive lethal phenotype, indicating that mutations G254I and F257R were not tolerated. Mutant I170EGR- and I275EGR-containing enzymes showed relatively normal Km and Vmax values, but they displayed a strong insensitivity to inhibition by vanadate. An I170EGR/I275EGR double mutant was more significantly perturbed showing a reduced Vmax and pronounced vanadate insensitivity. The I170EGR site within the putative alpha-helical stalk region was cleaved to a maximum of 10% by factor Xa under non-denaturing conditions resulting in a characteristic 81 kDa fragment, whereas the I275EGR site, near the end of the beta-strand region, showed about 30-35% cleavage with the appearance of a 70 kDa fragment. A I170EGR/I275EGR double mutant enzyme showed about 55-60% cleavage. The cleavage profile for the mutant enzymes was enhanced under denaturing conditions, but was unaffected by MgATP or MgATP plus vanadate. Cleavage at the I275EGR position had no adverse effects on ATP hydrolysis or proton transport by the H(+)-ATPase making it unlikely that this localized region of LOOP1 influences coupling. Overall, these results suggest that the local region encompassing I275EGR is accessible to factor Xa, while the region around I170EGR appears buried. Although there is no evidence for gross molecular motion at either site, the effects of multiple amino acid substitutions in these regions suggest that the LOOP1 domain is conformationally active, and that perturbations in this domain affect the distribution of conformational intermediates during steady-state catalysis.