Previously, we showed that ageing was associated with changes in the structure of the parotid gland and decreases in amylase secretion and cytosolic Ca²⁺ (Mahay et al. 2004). Since ageing is stress-related, we now investigate the effects of H₂O₂ on cytochrome c localisation in isolated parotid glands of young and aged male Wistar rats and amylase release and cytosolic Ca²⁺ in parotid glands of young (2-6 months) male Wistar rats. Animals were humanely killed and glands were isolated and either cut into small segments for the localisation of cytochrome c using immunocytochemistry or dissociated into acini for the measurements of amylase release and cytosolic Ca²⁺ signals (Pariente et al. 2000). All data are presented as mean ± SEM. Treatment of glands from 2-6 and 12 month old rats with H₂O₂ resulted in time-dependent (2 and 4 hr) increases in cytochrome c fluorescence, compared to untreated glands. Cytochrome c was localised more in the ductal regions of the gland compared to acinar tissues. Treatment of glands from 22 to 24 month old rats for the same times showed no visible increases in cytochrome c compared to the other two age groups. Basal amylase release in acini suspensions from rats 2-6 months was 5.47± 2.09 % of total (n = 8). Incubation of acini with either 0.5, 1 or 2 mM H₂O₂ resulted in significant (Student’s unpaired t test, p〈0.05) increases in amylase release compared to basal. Values for amylase release with 0.5, 1 and 2 mM H₂O₂ were 10.08 ± 0.90, 11.83 ± 1.93 and 10.36 ± 0.74 % of total (n = 8), respectively. Stimulation of acini with either ACh, NA or ISOP (10^-7 to 10^-4 M) resulted in dose-dependent release of amylase above basal. Pretreatment of acini with 1 mM H₂O₂ followed by the addition of either ACh, NA or ISOP resulted in significant (p〈0.05) decreases in amylase release compared to the responses obtained with secretagogues alone. [Ca²⁺]_i ratios (F340/F380) in fura-2 loaded parotid acinar cells isolated from 2-6 month old rats in the absence and presence of [Ca²⁺]_i was 0.845 ± 0.001, (n = 50 cells) 0.841 ± 0.003 (n = 50 cells) ratio units, respectively. Incubation of acinar cells with 1 mM H₂O₂ in either the absence or presence of [Ca²⁺]_o, resulted in a gradual increase in the Ca²⁺ signal. Application of 10^-5 M ACh after H₂O₂ incubation had no effect on these elevated Ca²⁺ signals. In contrast, ACh alone evoked a transient increase in Ca²⁺ signals followed by a plateau phase. Once, ACh-evoked Ca²⁺ signals were established, application of 1 mM H₂O₂ caused a further elevation in the Ca²⁺ signals. The results indicate that H₂O₂ can perturb cytochrome c levels, increase amylase release, alter Ca²⁺ homeostasis and antagonise the secretory response of ACh, NA or ISOP in the parotid gland.