Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed. Basal activities of AC2 and AC6 were compared at varying concentrations of Mg2+ or Mn2+ ions; AC2 had 15- and 10-fold greater activity than AC6, respectively. At 20 mM Mg2+, the Km values for ATP were 88 and 39 microM for AC2 and AC6, respectively, whereas their Vmax values were 281 and 11 pmol/mg protein.min. With 100 microM forskolin and either Mg2+ or Mn2+, the difference in activities between AC2 and AC6 was reduced to approximately 2-fold. Forskolin stimulated AC6 greater than 40-fold at 0.5-2 mM Mg2+, whereas AC2 was stimulated 4-6-fold. At 20 mM Mg2+, AC2 was stimulated 2-fold by forskolin, whereas AC6 was stimulated 18-fold. With Mg2+ alone, activities of AC2 and AC6 were not saturable up to 20 mM and yielded curvilinear Hofstee transformations. With forskolin, activities of both AC2 and AC6 were saturable by 10 mM Mg2+ and yielded linear Hofstee transformations. These data indicate that there are substantial differences in the basal enzymatic activities of adenylyl cyclase isoforms, due to differential regulation by Mg2+ ions rather than intrinsic catalytic capabilities. Thus the presence and relative abundance of adenylyl cyclase subtypes could greatly affect the resting cellular cAMP levels with consequent effects on important biological functions, such as differentiation and proliferation.