The effect of adenosine A(1) receptor activation on the ATP-induced increase in intracellular free Ca(2+) was studied in control and protein kinase C down-regulated Fisher rat thyroid (FRTL-5) cells. Long-term phorbol ester treatment, which leads to protein kinase C down-regulation, enhanced the ATP-evoked extracellular Ca(2+) influx. The increased Ca(2+) influx was antagonized by the adenosine A(1) receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). [3H]DPCPX binding studies revealed that phorbol ester-treatment increased the number of adenosine A(1) receptors. The adenosine A(1) receptor-mediated inhibition of the cyclic AMP formation was not affected by the increased receptor number. We conclude that the enhanced ATP-evoked Ca(2+) influx in protein kinase C down-regulated cells is mediated by adenosine formed by hydrolysis of ATP, and that this adenosine interacts with the increased number of A(1) receptors. The mechanism by which adenosine enhances Ca(2+) entry is not known. Thus, the larger number of adenosine A(1) receptors broadens the spectrum of adenosine A(1) receptor affected signaling systems in FRTL-5 cells.