OBJECTIVE The insulin tolerance test (ITT) is the established reference test for assessing the hypothalamo-pituitary-adrenal (HPA) axis. Various authorities, however, have suggested that the 250 microgram short ACTH stimulation test may be used to assess the HPA axis in place of the ITT in suspected hypopituitarism, although a number of other studies have suggested that the test may be unreliable in this setting. In this study, the ITT (or glucagon test) has been compared with the 250 microgram short ACTH stimulation test in patients with pituitary tumours pre-operatively and within 2 weeks of trans-sphenoidal hypophysectomy. METHODS An ITT, or a glucagon test when the ITT was contraindicated, and the 250 micrograms short ACTH stimulation test, were performed in all the patients before (an ITT in 18 and a glucagon test in three patients) and within 2 weeks after trans-sphenoidal hypophysectomy (an ITT in 16 and a glucagon test in five patients). METHODS Twenty-one patients with pituitary disorders (15 with acromegaly, one with a prolactinoma and five with non-functioning tumours) were studied; four had a microadenoma, two a mesoadenoma and 15 a macroadenoma. METHODS Serum cortisol was measured by radioimmunoassay. A normal response was defined as a rise in serum cortisol to 580 nmol/l or above for all the tests. RESULTS Before surgery, three of 18 patients (17%) had a discrepancy between the ITT and the 30 minute short ACTH stimulation test and one of three between the glucagon test and the 30 minute short ACTH stimulation test. Combined together, pre-operatively, four of 21 patients (19%) had discrepant results. In the early post-operative period, the discrepancy between ITT and the 30 minute short ACTH stimulation test was higher with four of 16 patients (25%) showing discordant results; one of the five patients tested had a discrepancy between the glucagon test and the 30 minute short ACTH stimulation test. On combining the tests, the post-operative discrepancy was five of 21 patients (24%). CONCLUSIONS The 250 microgram short ACTH stimulation test produces discordant results from the ITT in a clinically significant proportion of affected patients when assessing the HPA axis, although the rate of discordance varies according to the criteria used for normality for both the tests. We suggest that the ITT should remain the preferred test for assessing ACTH secretory capacity compared with the standard 250 microgram short ACTH stimulation test, both in the preoperative and in the early post-trans-sphenoidal hypophysectomy period.