OBJECTIVES: We evaluated the response to the thyrotropin-releasing hormone (TRH) stimulation test in very low-birth weight (VLBW) infants to elucidate the aetiology of transient hypothyroxinaemia of prematurity (THOP). DESIGN AND METHODS: We performed TRH stimulation tests on 43 VLBW infants. Subjects were divided into two groups; a THOP group (N = 11; basal TSH < 15 mU/L and basal FT4 ≤ 0.8 ng/dL) and a non-THOP group (N = 32; basal TSH < 15 mU/L and basal FT4 > 0.8 ng/dL). Basal FT4 and FT3 were measured before, and TSH (0, 30, 60, 90, 120 and 180 minutes) was measured after, the administration of TRH (7 µg/kg). We calculated the ratio of TSH 180 minutes to THS 0 minute as the primary outcome. We also collected data on T3 and rT3 in this study. RESULTS: In both groups, TSH 30 minutes values were the highest. However, the ratios of TSH 180 minutes to THS 0 minutes in the non-THOP group and the THOP group were (median [IQR]) 1.3 [1.0-1.7] and 3.0 [1.5-5.3] (P < .01). No significant differences were observed in T3 (1.0 [0.8-1.3] and 0.7 [0.4-0.7] ng/mL, P = .06). However, in the THOP group, rT3 was significantly lower than that of the non-THOP group (168.0 [148.1-197.0] and 92.9 [74.7-101.6] pg/mL, P < .01). CONCLUSIONS: The delayed decrease in the TSH concentration after the peak for the TRH tests and decreased levels of rT3 suggest that the main aetiology for THOP is suppression at the level of the hypothalamus, but not inactivation of peripheral thyroid hormone metabolism.