Subunits α, β, and γ of the F 1 -part of cyanobacterial F 0 F 1 -ATPase have been cloned into expression vectors. Overexpressed subunit β was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant α and γ subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the α and β subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F 1 -ATPase was assembled from its purified subunits α, β, γ, δ and ε. The reassembled enzyme reconstituted ATP synthesis in F 1 -depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.