Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.