In Synechocystis sp. PCC 6803 extracellular phosphate levels are relayed to the pho regulon via the SphS histidine kinase. In this cyanobacterium, the start codon of sphS has been assigned as a GUG, thereby predicting SphS to be a cytosolic protein lacking a putative N-terminal region found in the PhoR orthologue from Escherichia coli. Inspection upstream of sphS located an in-frame AUG positioned 47 codons in front of the putative GUG start. Alterations at either of the putative AUG or GUG start codons did not prevent transcription of sphS; however, up-regulation of alkaline phosphatase mRNA, or alkaline phosphatase activity, was not detected in response to phosphate-limiting conditions when the AUG was mutated. Alkaline phosphatase expression and activity serve as phenotypic markers for activation of the pho regulon. Therefore, the pho regulon had not been induced in these cells, whereas normal up-regulation was observed in strains carrying mutations at the GUG. These results show that the AUG codon, not the GUG codon, is the initiation site for sphS translation in Synechocystis sp. PCC 6803.