G protein-activated K(+) channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G(βγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R. Adenovirus-driven overexpression of the A(1)-R caused an increase in current induced by adenosine (I(K(Ado))), whereas the M(2)-R-activated current (I(K(ACh))) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M(2)-R resulted in a reduction in I(K(ACh)) without affecting I(K(Ado)), arguing against a competition of the two receptors for common signaling complexes. The negative effect of A(1)-R overexpression on I(K(ACh)) was reduced by the A(1)-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I(K(ACh)) and I(K(Ado)), suggesting common pathways of A(1)-R and M(2)-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I(K(ACh)) and I(K(Ado)). Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A(1)-R in an autocrine fashion. Chronic activation of A(1)-R causes parallel downregulation of both A(1)-R and M(2)-R.