Cultured astrocytes prepared from the newborn rat cerebral cortex were challenged with a variety of purine and pyrimidine nucleotides and changes in intracellular Ca2+ concentration ([Ca2+]i) assessed. ATP, ADP, 2-methylthioadenosine triphosphate (2-MeSATP) and the pyrimidine nucleotide uridine triphosphate (UTP) produced Ca2+ transients which were characterized by an initial spike which decayed rapidly to a secondary plateau phase. Concentration response curves for these ligands gave the following rank order of potency: 2-MeSATP > ADP > ATP > UTP. Analysis of the number of cells responding to these nucleotides revealed that 2-MeSATP, ATP and ADP were effective in all cell fields examined whilst UTP generated a response in only 40%. In some experiments cells were challenged with various combinations of agonists. In those fields where ATP, 2-MeSATP and UTP were effective, the transients elicited by combined additions of these agonists were essentially the same as those produced by the agonists alone. Of the other nucleotides tested, uridine diphosphate was effective in only 24% of cell fields examined, moreover, its transient appeared to lack an initial spike phase. Guanosine triphosphate (GTP), cytidine triphosphate (CTP) and deoxythymidine triphosphate (TTP) were found to increase [Ca2+]i in 75%, 47% and 37% of cell fields respectively and the transients generated appeared to lack a secondary plateau phase. The responses to GTP, CTP and TTP also displayed rapid desensitization, a situation never encountered with ligands such as ATP.