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To determine the optimal observation period (OBP) in adults with a clinical diagnosis of brain death (BD) using electroencephalography (EEG) or computerized tomography angiography (CTA). We conducted a retrospective observational analysis of adult patients with a diagnosis of BD from January 2000 to February 2017. The optimal OBP was defined as the minimum time interval from the first complete clinical neurological examination (CNE) that ensures that neither a second CNE nor any ancillary test (AT) performed after this period would fail to confirm BD. The study sample included 447 patients. In the supratentorial group, the first AT confirmed BD in 389 cases (98%), but in 8 (2%) cases the complementary test was incongruent. In this group, 8 of 245 patients in whom the first AT was carried out within the first 2 h after a complete CNE had a non-confirmatory test of BD versus none of 152 in whom the first AT was delayed more than 2 h (3.0% vs 0.0%; p = 0.026). In the infratentorial group, we found a higher probability of obtaining a first non-confirmatory AT of BD (34% vs 2%; p = 0.0001) and an OBP greater than 32.5 h was necessary to confirm a BD diagnosis. We found important differences in the confirmation of BD diagnosis between primary supratentorial and infratentorial lesion, and identified an optimal OBP of 2 h in patients with supratentorial lesions. By contrast, in primary posterior fossa/infratentorial lesions, the determination of an optimal OPB remains less accurate and hence more challenging.