Urine samples from 10 normal adult males were analyzed for T4-binding globulin (TBG) employing a sensitive and specific RIA for serum TBG. Urine TBG was detected at a concentration of 1.74 +/- 0.87 microgram/100 ml (mean +/- SD). The excretion rate was 13.2 +/- 6.5 microgram/g creatinine. There was no correlation between the daily excretion rate of TBG and the total urinary protein (r = 0.52). Analyses of this T4-binding protein by gel filtration and on both reverse flow paper and gel electrophoresis demonstrated similar characteristics to those of serum TBG and purified TBG. The maximum T4-binding capacity of urine TBG, determined by reverse flow paper electrophoresis and equilibrium dialysis, was similar to that of serum TBG. The association constant for T4 of urinary TBG (0.46 +/- 0.20 X 10(10) M-1), determined by equilibrium dialysis, was significantly less (P less than 0.01) than that of serum TBG at 1.43 +/- 0.23 X 10(10) M-1). The Scatchard analysis of the urinary data demonstrated two T4-binding sites, one of high affinity consistent with TBG and one of low affinity consistent with albumin. It is concluded that the TBG present in urine has the major physical and biochemical features of the intact molecule, but that minor conformational and/or charge alterations occurred during renal excretion. Interpretation of data on thyroid hormones in normal urine should take the presence of TBG into consideration.