The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H2O2) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca2+]i) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H2O2 had little or no effect on amylase secretion compared to basal level. Combining H2O2 with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca2+]i comprising an initial peak followed by a plateau. In a Ca2+-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca2+]i. H2O2 alone evoked a gradual and dose-dependent increase in [Ca2+]i. Combining H2O2 with CCh resulted in a decrease in [Ca2+]i compared to the effect of CCh alone. In a Ca2+-free medium, H2O2 still evoked a small increase in [Ca2+]i, but this response was less compared to the results obtained with H2O2 in normal [Ca2+]0. Combining H2O2 with CCh resulted in only a small transient increase in [Ca2+]i. Following CCh stimulation, H2O2 application resulted in a large increase in [Ca2+]i in normal [Ca2+]0. This effect of H2O2 was partially abolished in a nominally free Calcium medium containing EGTA. H2O2 can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H2O2 can also mobilise Ca2+ from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H2O2 may be due to its activity to induce apoptosis in the parotid gland, since H2O2 can stimulate the activity of caspase-3, a marker of cellular apoptosis.