The present study presents the first evidence for P₂Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca²⁺](i)) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca²⁺ in the solutions ATP increased [Ca²⁺](i). The increase in [Ca²⁺](i) was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca²⁺](i) caused by the various compounds. The rise in [Ca²⁺](i) was predominantly monophasic at low ATP concentrations (below 100 μM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca²⁺ in the extracellular solution ATP caused Ca²⁺ release from intracellular stores. This could be inhibited by pre- treatment of the tissue with 1 μM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca²⁺](i) although the plateau level of the [Ca²⁺](i) response was higher with this P₂Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P₂Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.