The vestibular cell type affected by congenital hypothyroidism (CH) was investigated by measuring the activity of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT), synthesizing enzymes of putative afferent (GABA) and efferent (acetylcholine, ACh) neurotransmitters and thus, respectively, hair cell I and II (HC-I, HC-II), and efferent terminal (ET) marker enzymes, in vestibular homogenates of control, congenitally hypothyroid rats (CHR) and in thyroxine-replaced CHR (CHR-T4) whose postnatal age ranged from 20 to 60 days old. In the vestibule, CH-II and its efferent cholinergic contacting bouton mature prior to thyroid function whereas HC-I-differentiation and its efferent synapse arrival are the latest events in vestibular maturation. Therefore, a differential effect of CH upon GAD and ChAT in CHR could be anticipated. In control rats as in CHR the magnitude of GAD was the same with time starting on the 20th day. In CHR, ChAT gradually diminished beginning on day 28 to become 45% decreased with respect to control on the 60th postnatal day. Prevention of ChAT decrease in CHR by early administration of thyroxine (T4), a striking diminution of T4 and triiodothyronine (T3) in CHR serum and a normal level of these hormones found in CHR-T4 corroborated thyroid involvement. These results confirm the preference of hypothyroidism to affect cholinergic cell types (or compartments) of late maturation (HC-I-containing ET and hence 45% ChAT decrease) leaving HC-I, HC-II and HC-II-connecting ET untouched, supported by a 55% remanent ChAT and a constant GAD activity regardless of time and treatment.