Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by a-chymotrypsin. The purification (26000-fold from the particulate fraction or 125 000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5 -10% and a final specific activity of 19 pmol cyclic AMP formed mg protein-' min-' at 30°C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30"C, 20°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4°C. The purified enzyme exhibited a K, value similar to that of the native enzyme (K, = 1.4 mM).Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Bid. Chem. 257, 10818-108261, gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.Adenylate cyclase from mammalian cells comprises at least three components [l]. First, hormonal receptors responsible for activation or inhibition of adenylate cyclase activity. Secondly, the two coupling N (or G) proteins, a stimulatory Ns, and a inhibitory Ni. These N proteins have been purified in different tissues to homogeneity: both are a, p, y heterotrimers; their p subunits appear to be identical, but they differ in their a subunit. To date, the only components whose primary structure has been elucidated by direct cloning procedures are the a, and ai components [2-41. The third component is the catalytic subunit, which catalyzes the conversion of ATP in cyclic AMP in the presence of divalent cations.