Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell lines with one gene inactivated were used to characterize the other guanylyl cyclase (i.e. GCA in sgc ؊ null cells and sGC in gca ؊ null cells). Despite the different topologies, the enzymes have many properties in common. In vivo, extracellular cAMP activates both enzymes via a G-protein-coupled receptor. In vitro, both enzymes are activated by GTP␥S (K a ؍ 11 and 8 M for GCA and sGC, respectively). The addition of GTP␥S leads to a 1.5-fold increase of V max and a 3.5-fold increase of the affinity for GTP. Ca 2؉ inhibits both GCA and sGC with K i of about 50 and 200 nM, respectively. Other biochemical properties are very different; GCA is expressed mainly during growth and multicellular development, whereas sGC is expressed mainly during cell aggregation. Folic acid and cAMP activate GCA maximally about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic stress strongly stimulates sGC but has no effect on GCA activity. Finally, GCA is exclusively membrane-bound and is active mainly with Mg 2؉ , whereas sGC is predominantly soluble and more active with Mn 2؉ .cAMP and cGMP are important signaling molecules. In prokaryotes, cAMP regulates gene expression. Cyanobacteria contain high levels of cGMP relative to other bacteria, but their function as intracellular signaling molecules is not well understood (1). In eukaryotes, cAMP and cGMP regulate enzyme activities, channel activity, and gene expression, mainly via cAMP-and cGMP-dependent protein kinase (2, 3). A large and complex family of adenylyl cyclase (AC) 1 and guanylyl cyclase (GC) is responsible for the synthesis of cAMP and cGMP (4, 5).The crystal structure of mammalian AC (6, 7) suggests that the core of the enzyme consist of two cyclase domains that are associated in an antiparallel manner. In metazoan, four cyclase subgroups are recognized (4, 5, 8): 1) the 12-transmembrane adenylyl cyclase is composed of two different cyclase domains and is regulated by G-proteins; 2) The single-transmembrane guanylyl cyclase contains one cyclase domain and functions as a homodimer, and GC activity is stimulated by extracellular peptides; 3) The nitric oxide-sensitive soluble guanylyl cyclase is a complex of two different proteins with one cyclase domain each; 4) The recently discovered soluble adenylyl cyclase (sAC) from rat and human possesses two cyclase domains, which share the highest degree of identity with bacterial adenylyl cyclases (9).In the social amoeba Dictyostelium, cGMP is implicated as one of the second messengers for chemotaxis (10), although its precise role is not known. On the other hand, cAMP can act as both first and second messenger (11, 12). As first messenger, cAMP induces chemotaxis, cAMP signal relay, and gene expression. Dictyostelium possesse...