1. The changes in the free cytosolic Ca2+ concentration ([Ca2+]i) in response to agonists of P2-purinoceptors were studied in myocytes isolated from the longitudinal muscle layer of different regions of the rat gastrointestinal tract (stomach, jejunum, ileum, caecum and colon). [Ca2+]i was estimated by emission from the fluorescent dye, indo-1. 2. ATP and the P2Y-purinoceptor agonist, 2-methylthio-ATP (2-MeSATP), transiently increased [Ca2+]i in single myocytes from all segments of the gastrointestinal tract, whereas alpha,beta-methylene-ATP, a P2x-purinoceptor agonist, had no effect. 3. The rise in [Ca2+]i induced by ATP and 2-MeSATP was maintained in Ca(2+)-free solution but was abolished by depletion of the intracellular store with thapsigargin (1 microM). 4. Single myocytes from stomach, caecum and colon also responded to UTP by a transient increase in [Ca2+]i. 5. Individual myocytes responded to ATP, 2-McSATP and UTP in a nearly all-or-nothing manner. The increasing of agonist concentration enhanced the number of responding cells but did not increase the amplitude of the [Ca2+]i rise. 6. These results suggest that myocytes from the longitudinal layer of gastrointestinal muscle do not possess functional P2x-purinoceptors and that agonists of P2Y and P2U-purinoceptors induced a rise in [Ca2+]i, probably via an all-or-nothing mobilization of Ca2+ from intracellular stores.