OBJECTIVE We compared the performance of tandem mass spectrometry versus immunoassay for measuring thyroid hormones in a diverse group of inpatients and outpatients. METHODS Thyroxine (T4), triiodothyronine (T3), free thyroxine (FT4), and free triiodothyronine (FT3) were measured by liquid chromatography tandem mass spectrometry and immunoassay in 100 patients and the two assays were compared. RESULTS T4 and T3 values measured by the two different assays correlated well with each other (r=0.91-0.95). However, the correlation was less good at the extremes (r=0.51-0.75). FT4 and FT3 concentrations measured by the two assays correlated less well with each other (r=0.75 and 0.50 respectively). The studied analytes had poor inverse correlation with the log-transformed TSH values (r=-0.22-0.51) in the population as a whole. The strongest correlations were seen in the groups of outpatients (r=-0.25-0.61). The weakest degree of correlation was noted in the inpatient group, with many correlations actually being positive. CONCLUSIONS The worst between-assay correlation was demonstrated at low and high hormone concentrations, in the very concentration ranges where accurate assay performance is typically most clinically important. Based on the lesser susceptibility of mass spectrometry to interferences from conditions such as binding protein abnormalities, we speculate that mass spectrometry better reflects the clinical situation. In this mixed population of inpatients and outpatients, we also note failure of assays to conform to the anticipated inverse linear relationship between thyroid hormones and log-transformed TSH.