Cyanobacterial genomes harbour two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants, respectively. Genes gap1 and gap2 of the unicellular cyanobacterium Synechocystis sp. PCC 6803 were cloned and sequenced and subsequently inactivated by insertional mutagenesis to understand their metabolic functions. We obtained homozygous gap1- mutants which have lost the capacity to grow on glucose under dim light while growth on organic acids as well as photosynthetic growth under CO2 and high light is not impaired. Homozygous gap2- mutants show the reciprocal phenotype. Under dim light they only grow on glucose but not on organic acids nor do they survive under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from wild type and mutant cells show that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD-specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases three- to fivefold after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild-type cells, play distinct key roles in catabolic and anabolic carbon flow, respectively. While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which the catabolic activity of Gap2 seems to be repressed by a specific low-molecular-weight inhibitor.