Although many in vitro studies demonstrated that thiopental sodium (TPS) is a promising neuroprotective agent, clinical attempts to use TPS showed mainly unsatisfactory results. We investigated the neuroprotective effects of TPS against hypoxic insults (HI), and the responses of the neurons to L-glutamate and acetylcholine application. Neurons prepared from El 7 Wistar rats were used after 2 weeks in culture. The neurons were exposed to 12-h HI with or without TPS. HI-induced neurotoxicity was evaluated morphologically. Moreover, we investigated the dynamics of the free intracellular calcium ([Ca2+]i) in the surviving neurons after HI with or without TPS pretreatment following the application of neurotransmitters. TPS was neuroprotective against HI according to the morphological examinations (0.73 +/- 0.06 vs. 0.52 +/- 0.07, P = 0.04). While the response to L-glutamate was maintained (0.89 +/- 0.08 vs. 1.02 +/- 0.09, P = 0.60), the [Ca2+]i response to acetylcholine was notably impaired (0.59 +/- 0.02 vs. 0.94 +/- 0.04, P < 0.01). Though TPS to cortical cultures was neuroprotective against HI morphologically, the [Ca2+]i response not to L-glutamate but to acetylcholine was impaired. This may partially explain the inconsistent results regarding the neuroprotective effects of TPS between experimental studies and clinical settings. (C) 2016 Elsevier B.V. All rights reserved.