The purpose of this study was to determine the influence of testosterone, the male sex hormone, on β-adrenergic agonist-induced amylase secretion from rat parotid glands. Isoprenaline (isoproterenol)-induced amylase secretion was measured in vitro from the parotid glands of control and castrated rats with and without testosterone replacement. The isoprenaline-induced amylase release was reduced in parotid glands from castrated rats compared to controls. The reduction of amylase release by isoprenaline in parotid glands of castrated rats, could be reversed by administration of testosterone. Furthermore, β-adrenergic receptor density and the level of isoprenaline-evoked cAMP in parotid glands from castrated rats was lower compared to intact rats. Using SQ-22536 (an adenylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analogue) and verapamil (a calcium channel blocker), we conclude that the impairment of amylase release from parotid glands after castration was not related to either adenylyl cyclase activity or cAMP accumulation. Amylase release from the parotid glands of castrated rats appears to be mediated by an increase in calcium ion influx.