cotA of Synechocystis sp. strain PCC6803 is a gene involved in light-induced proton extrusion (A. Katoh, M. Sonoda, H. Katoh, and T. Ogawa, J. Bacteriol. 178:5452-5455, 1996). There are two possible initiation codons in cotA, and either long (L-) or short (S-) cotA encoding a protein of 440 or 247 amino acids could be postulated. To determine the gene size, we inserted L-cotA and S-cotA into the genome of a cotA-less mutant (M29) to construct M29(L-cotA) and M29(S-cotA), respectively. M29(L-cotA) showed essentially the same net proton movement profile as the wild type, whereas no light-induced proton extrusion was observed with M29(S-cotA). Two kinds of antibodies were raised against partial gene products of the N- and C-terminal regions of L-cotA, respectively, fused to glutathione S-transferase expressed in Escherichia coli. Both antibodies cross-reacted with a band at 52 kDa in both cytoplasmic and thylakoid membrane fractions of the wild-type cells. The same cross-reacting band was present in the membranes of M29(L-cotA) but not in M29 or M29(S-cotA). These antibodies cross-reacted more strongly with the cytoplasmic membrane fraction than with the thylakoid membrane fraction. The antibody against NrtA, a nitrate transporter protein present only in the cytoplasmic membrane, also cross-reacted with the thylakoid membrane fraction strongly. Based on these results we concluded that CotA of 440 amino acids (51 kDa) is located in the cytoplasmic membrane. Whether CotA is absent in the thylakoid membrane remains to be solved.