A two-component signal transduction system involved in phosphate-sensing system, SphS-SphR system, was reported for an actual start codon of sphS and a possible interaction between a SphS kinase protein and a negative regulator SphU in 2007. In this study the requirement of the N-terminus of SphS and the phosphate-specific transport (Pst) system for regulation of the SphS-SphR system has been shown. A putative membrane-spanning region between amino acids Ile-4 and Ile-19 of SphS is required for activation of the SphS–SphR phosphate-sensing two-component system under phosphate-limiting conditions as mutants lacking this region are unable to express alkaline phosphatase, the Pho regulon expression indicator, even grown under phosphate-limiting conditions or the negative regulator SphU is inactivated. Internal deletion of 8 amino acids of the putative membrane-spanning region between amino acids Ile-8 and Gly-15, however, leads to constitutive alkaline phosphatase expression. This constitutive expression is not related to protein length as deletions of 4, 6 and 9 amino acids in other region do not affect the regulation of the SphS–SphR phosphate-sensing system. Furthermore, it seems regulation of SphS-SphR system does not need specific amino acids around Ile-8 and Gly-15, alanine substitutions in some amino acids do not alter SphS-SphR system activity. In addition, two Pst systems in Synechocystis sp. PCC 6803 have been deleted, designated ΔPst1 and ΔPst2 strains. Our results show that the Pst1 system, but not the Pst2 system, is required for suppression of the Pho regulon under phosphate-sufficient conditions. Deletion of the pst1 operon and disruption of the membrane-spanning domain may both target the same control mechanism since constitutive alkaline phosphatase activity is similar in the double and single mutants. Both Pst1 and Pst2 systems are active and can transport phosphate in the amount that is sufficient for cell growth and pigment biosynthesis, compared to wild type. The study of kinetics of phosphate uptake showed a Km of 0.13, 5.16 and 80.67 µM in ∆Pst1, ∆Pst2 and wild type, respectively. In addition, Vmax values of 0.18, 2.17 and 3.12µmol/ min/ mg of chlorophyll a were obtained for the ΔPst1, ΔPst2 and wild-type strains, respectively. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects.