Adenosine A 1 receptors induce an inhibition of adenylyl cyclase via G-proteins of the G i/o family. In addition, simultaneous stimulation of A 1 receptors and of receptor-mediated activation of phospholipase C (PLC) results in a synergistic potentiation of PLC activity. Evidence has accumulated that G␤␥ subunits mediate this potentiating effect. However, an A 1 receptormediated increase in extracellular glutamate was suggested to be responsible for the potentiating effect in mouse astrocyte cultures. We have investigated the synergistic activation of PLC by adenosine A 1 and ␣ 1 adrenergic receptors in primary cultures of astrocytes derived from different regions of the newborn rat brain. It is reported here that (1) adenosine A 1 receptor mRNA as well as receptor protein is present in astrocytes from all brain regions, (2) A 1 receptor-mediated inhibition of adenylyl cyclase is of similar extent in all astrocyte cultures, (3) the A 1 receptor-mediated potentiation of PLC activity requires higher concentrations of agonist than adenylyl cyclase inhibition and is dependent on the expression level of A 1 receptor, and (4) the potentiating effect on PLC activity is unrelated to extracellular glutamate.Taken together, our data support the notion that ␤␥ subunits are the relevant signal transducers for A 1 receptor-mediated PLC activation in rat astrocytes. Because of the lower affinity of ␤␥, as compared with ␣ subunits, more ␤␥ subunits are required for PLC activation. Therefore, only in cultures with higher levels of adenosine A 1 receptors is the release of ␤␥ subunits via G i/o activation sufficient to stimulate PLC. It is concluded that variation of the expression level of adenosine A 1 receptors may be an important regulatory mechanism to control PLC activation via this receptor.