Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M2AChR pharmacological profile. Therefore, a novel Ca2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [3H]cGMP and [3H]cAMP exhibiting a higher affinity as Km (mM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC50 of 4.9 and 4.6 mM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaMactivated PM-bound PDE1A, linked to M2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed. Abbreviations: ASM: Airway smooth muscle; BTSM: Bovine Tracheal Smooth Muscle; Benzamidine: amidinobenzene hydrochloride; CaM: calmodulin; CCh: carbamylcholine; cAMP: cyclic adenosine 30, 50-monophosphate; cGMP: cyclic guanosine 30,50- monophosphate; 4-DAMP: 4-diphenylacetoxy-N-methylpiperidine; DTT: 1:4-dithiothreitol; PDE1A: phosphodiesterase 1A; PDE1B: phosphodiesterase tipo 1B; PDE1C: phosphodiesterase tipo 1C; PMSF: phenylmethylsulphonylfluoride; IBMX: isobutyl-methylxanthine; GPCR: G protein-coupled receptors; mAchR or MAchR: muscarinic acetylcholine receptor; M2AChR: M2 muscarinic receptor; M3AChR: M3 muscarinic receptor; NO-sGC: NO sensitive soluble guanylyl cyclase; NPR-GC: Natriuretic Peptides Receptor guanylyl cyclase; TCA: Trichloroacetic acid; Vinpocetine: (3a: 16b)-eburnamenine-14-carboxylic ethyl ester acid