Changes in intracellular free calcium ([Ca++]i) play an important role in a variety of biochemical reactions that lead to cellular responses such as proliferation and differentiation. The response of [Ca++]i to extracellular nucleotides (ATP, UTP, ITP, and AMP-PNP) was determined in individual canine keratinocytes using the fluorescent probe fura-2 and digital video fluorescence imaging microscopy. In the presence of 1.8 mM extracellular Ca++, 100 and 500 microM ATP caused a rapid (less than 9 sec) three- to twelvefold rise in [Ca++]i above resting levels of 50-150 nM followed by occasional fluctuations. Small responses were elicited with doses as low as 0.1 microM ATP. The response of cells stimulated with 500 microM ATP in Ca(++)-free medium was characterized by 1.5 to 3 times rapid initial peak followed by a decrease of [Ca++]i below resting levels. Loss of response occurred in the majority of keratinocytes preincubated for 30 min in Ca(++)-free medium. UTP was as effective as ATP in stimulating rises in [Ca++]i in keratinocytes. Smaller elevations in [Ca++]i up to four- to fivefold resting levels were noted with 100 microM AMP-PNP or 500 microM ITP. Desensitization of cells was demonstrated when a second stimulation followed the primary ATP or UTP treatment. These results are suggestive of the presence of purinergic receptors in the cytoplasmic membrane of canine keratinocytes. Experiments using the calcium channel blocker lanthanum suggest that ATP-induced initial rises and sustained levels of [Ca++]i are dependent on the release of Ca++ from intracellular stores. These intracellular Ca++ stores appear to be rapidly depleted after removal of extracellular calcium ([Ca++]e), thereby abolishing ATP-induced [Ca++]i increases.