The H+-translocating pyrophosphatase (H+-PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H+-PPase expressed in Escherichia coli using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N- and C-terminal segments. Thirty-six of the mutant enzymes retained both pyrophosphatase and H+-translocating activities. Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S. coelicolor H+-PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus. We propose that the basic structure of H+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.