ATP synthase is unique with respect to its high efficiency and reversibility in interconversion between chemical energy and mechanical work. Although rotary dynamics of F1, ATP-driven motor part of ATP synthase, has been intensively studied, the dynamics and transporting activity of the whole complex of ATP synthase are poorly studied at single-molecule level, mainly due to technical difficulties to handle the protein reconstituted in lipid bilayer. In this presentation, I will introduce two novel single-molecule analysis systems for ATP synthase working in membrane. First one is a supported membrane system into which we reconstituted ATP synthase molecules. When caged-proton molecules immersed within the thin layer between the supported membrane and solid substrate (coverslip) was selectively uncaged, proton-motive-force was transiently formed to drive the rotation of ATP synthase in the ATP synthesis direction. The molecules showed biased Brownian rotation with 120 degree steps, showing that the kinetic bottleneck is a catalytic event on F1 motor having a pseudo-three-fold symmetry under the conditions where pmf is mostly balanced with free energy of ATP hydrolysis. More recently, we successfully developed an arrayed lipid bilayer chambers (ALBiC) system; arrayed femtoliter chambers, each sealed with lipid bilayer patch. When stochastically reconstituted with a single molecule of ATP synthase, some chambers showed distinctive proton-transporting activity, measured with pH sensitive dyes. From the time-dependent pH change, the proton-pumping activity of ATP synthase was determined to be about 30 protons per second that is in good agreement with previous estimated value in biochemical assays.