Thyroid hormone (TH) deficiency during perinatal life causes a multitude of functional and morphological deficits in the brain. In rats and mice, TH dependency of neural maturation is particularly evident during the first 1–2 weeks of postnatal development. During the same period, synaptic transmission via the inhibitory transmitters glycine and GABA changes from excitatory depolarizing effects to inhibitory hyperpolarizing ones in most neurons [deploarizing–hyperpolarizing (D/H) shift]. The D/H shift is caused by the activation of the K+–Cl− co-transporter KCC2 which extrudes Cl− from the cytosol, thus generating an inward-directed electrochemical Cl− gradient. Here we analyzed whether the D/H shift and, consequently, the onset of inhibitory neurotransmission are influenced by TH. Gramicidin perforated-patch recordings from auditory brainstem neurons of experimentally hypothyroid rats revealed depolarizing glycine effects until postnatal day (P)11, i.e. almost 1 week longer than in control rats, in which the D/H shift occurred at ∼P5–6. Likewise, until P12–13 the equilibrium potential EGly in hypothyroids was more positive than the membrane resting potential. Normal EGly could be restored upon TH substitution in P11–12 hypothyroids. These data demonstrate a disturbed Cl− homeostasis following TH deficiency and point to a delayed onset of synaptic inhibition. Interestingly, immunohistochemistry demonstrated an unchanged KCC2 distribution in hypothyroids, implying that TH deficiency did not affect KCC2 gene expression but may have impaired the functional status of KCC2. Hippocampal neurons of hypothyroid P16–17 rats also demonstrated an impaired Cl− homeostasis, indicating that TH may have promoted the D/H shift and maturation of synaptic inhibition throughout the brain.