A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the alpha- as well as the beta-subunit of the gastric H,K-ATPase. Sf9 cells infected with this virus exhibited a K(+)- and SCH 28080-sensitive ATP-dependent phosphorylation capacity in purified Sf9 membranes similar to native H,K-ATPase. This activity was not present in control membranes containing only one of the two H,K-ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H,K-ATPase.