Using a specific radioimmunoassay (RIA) for deamido TRH (TRH-OH), TRH-OH immunoreactivity (IR) was detected in the urine of 54% of a group of normal subjects and patients with thyroid disease. Human urine TRH-OH IR eluted in the position of synthetic TRH-OH on Sephadex G-10 gel filtration (GF). In contrast, urine TRH IR, as determined by a specific TRH RIA, did not elute with synthetic TRH. HU-TRH was prepared for studies on its crossreactivity in the TRH-OH RIA. HU-TRH is the term given to a previously described substance(s) which is obtained from human urine by immunoabsorption to anti-TRH antiserum, is reactive in the TRH RIA, and shows non-identity with synthetic TRH on GF. HU-TRH was not reactive in the TRH-OH RIA. These studies suggest that the putative TRH metabolite, deamido TRH, is present in human urine and that HU-TRH is neither TRH-OH or as closely related structurally to TRH-OH as it is to TRH.