In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526-S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of beta-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.