Receptor-mediated G protein-linked adenylyl cyclase systems are universal signal transducers. We exploited the essential role of this cascade in Dictyostelium development to screen for random mutations in the catalytic component, ACA. This enzyme is activated by G protein betagamma-subunits acting in concert with a novel cytosolic regulator, CRAC. By suppression of the CRAC-null phenotype, we isolated constitutively active versions of the enzyme that require neither exogenous stimuli nor internal regulators. One mutant displayed a 15-fold increase in its Vmax. It harbors a single amino acid substitution (L394S) affecting a conserved residue located in the first cytoplasmic loop near the N-terminal hydrophobic domain of ACA. The screening procedure can be adapted for isolation of constitutive mutations in mammalian adenylyl cyclases.