TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration with little tubular resorption. The possible role of the prostate as a source of urinary TRH-LI was evaluated in 11 men with prostate cancer, showing a 25% decrease in urinary TRH-LI excretion after prostatectomy (0.19 +/- 0.02 vs. 0.15 +/- 0.01 ng/mumol creatinine, mean +/- SEM). However, TRH-LI was similar in spontaneously voided urine and in urine obtained through a nephrostomy cannula from 16 patients with unilateral urinary tract obstruction (0.15 +/- 0.01 vs. 0.14 +/- 0.01 ng/mumol creatinine). These data indicate that: 1) TRH-LI in human serum represents largely < EEP-NH2, which is cleared by renal excretion; 2) part of urinary < EEP-NH2 is derived from prostatic secretion into the blood and not directly into urine; and 3) urinary < EEP-NH2 can be used as marker for carcinoid tumors.