Salivary cortisol reflects the free fraction of serum cortisol. Monitoring salivary cortisol may be a promising alternative method for assessing serum cortisol in some clinical situations. We aimed to compare the reliability of salivary vs. serum cortisol during ACTH test. 84 subjects (mean age 63.2; 24-89 years; n=66 males) suspected for adrenocortical insufficiency underwent an ACTH test. Patients were divided based on peak serum cortisol into hypocortical group with cortisol <500 nmol/l and to reference group cortisol >500 nmol/l. Median serum cortisol levels in reference group were 445, 766, and 902 nmol/l at 0, 30, and 60 minutes, respectively, and in hypocortical group were 256, 394, and 453 nmol/l. Median salivary cortisol levels were 19.02, 40.02, and 62.1 nmol/l in reference group, and 9.60, 14.08, and 13.28 nmol/l in hypocortical group. Obtained values showed good correlation between serum and salivary cortisol (p<0.0001). The percentage of explained variability R(2) (coefficient of determination for linear model) representing a measure of agreement between experimental values and predictions for repeated measures ANOVA, was significantly higher (p=0.021) for serum cortisol (R2=93.4 %) when compared to the salivary cortisol (R2=89.3 %). A stronger discriminating power of serum versus salivary cortisol suggests that it seems to be slightly, but statistically significantly more appropriate marker of adrenocortical reserve in ACTH test.