Iodothyronines were measured by RIA in concentrated human cerebrospinal fluid (CSF). In measurements performed in one laboratory at a 4-fold concentration of CSF, rT3 was detected in all 30 patients in whom the measurement was attempted and averaged 15.1 +/- 2.8 ng/dl (mean +/- SE). IN an independent laboratory, rT3 measurements at this 4-fold concentration were performed on 11 different samples and averaged 9.3 +/- 0.9 ng/dl, while at a 40-fold CSF concentration, rT3 averaged 13.1 +/- 0.72 ng/dl. CSF T3 values were detected in only 4 of 30 patients as a 4-fold concentration and averaged 8.2 +/- 3.1 ng/dl, while in pooled CSF at a 40-fold concentration, 4 of 4 samples were detectable and averaged 2.6 +/- 0.4 ng/dl. Levels of T4 could only be detected in CSF concentrated 40-fold and averaged 0.22 +/- 0.04 microgram/dl. Values for the percent dialyzable thyronines were 0.44 +/- 0.03% for T4, 4.39 +/- 0.29% for T3, and 0.91 +/- 0.04% for rT3. TSH was detected in CSF concentrated 4-fold, averaging 0.43 +/- 0.05 microunits/ml; in an independent assay with CSF concentrated 40-fold, TSH averaged 0.06 +/- 0.02 microunits/ml. Thyroid binding globulin was undetectable (less than 0.1 mg/dl). We have confirmed the presence of thyroid hormones (T4 and T3) in human CSF and have shown that rT3 is present as well. The role these hormones play in the development, function, and nourishment of the central nervous system and the role the CSF plays in the transport of these hormones into the central nervous system remains to be determined.