We describe the clinical application of a radioimmunoassay combined with fast protein liquid Chromatography (FPLC) for measuring TRH immunoreactivity (TRH-IR) in blood samples extracted previously with methanol or with Sep Pak C18 cartridges. Sensitivity of the RIA was 3 fmol/tube, displacement at 50% B/B0 was achieved by 55 fmol of unlabelled TRH. Our specific antibody K2B7 (km = 2.2 fM) showed no cross reaction with other peptides. No difference was observed between the mean values of TRH-IR in 19 euthyroid, 22 hyperthyroid, 18 hypothyroid and 10 hypophysectomised patients (45 +/- 17.8, 58 +/- 30, 40 +/- 22 and 36 +/- 12 fmol/ml, mean +/- SD, respectively), whereas TRH-IR was significantly lowered (p less than 0.05) in 6 euthyroid pancreatectomised patients (21 +/- 5 fmol/ml). The reversed phase FPLC analysis of the TRH-IR presented in the methanol extracts was shown to have the same retention time as synthetic TRH. TRH could not be measured in unextracted blood samples. TRH added in preincubated (60 min, at 37 C), before extraction, blood samples showed a loss of 83.4% of immunoreactivity. Our results demonstrate that this method is able to detect TRH-IR in human blood by whole methanol extraction and/or by Sep Pak C18 cartridges extraction. Furthermore the findings suggest that the main source of circulating TRH-IR may be of extrahypothalamic (pancreatic?) origin and that the basal peripheral TRH levels are not involved or they do not clearly represent a pathological condition.