Type V adenylyl cyclase (ACV) belongs to the family of Ca
2+
- inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the α subunit of the stimulatory G protein G
s
(G
sα
) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the α subunit of the inhibitory G protein G
i
(G
iα
) inhibited all forms of the enzyme, indicating that the regions for G
sα
and G
iα
interaction are preserved in the soluble forms. Ca
2+
inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G
sα
and forskolin obliterated the Ca
2+
-mediated inhibition of the full-length and C1-C2 forms of ACV. In
45
Ca
2+
overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca
2+
. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound
45
Ca
2+
. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G
sα
and G
iα
is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca
2+
and inhibition of enzyme activity.