Objective: Search for germline mutations in the thyroxine-binding globulin (TBG) gene of two unrelated Portuguese females of Caucasian origin in whom the diagnosis of TBG deficiency was suspected because of suppressed TSH despite marginally low total thyroxine and tri-iodothyronine. Design and Methods: Screening for germline mutations was conducted by non-radioactive PCR-SSCP analysis. The variants documented by this approach were characterized by sequencing. Moreover, in order to define whether they were mutations or polymorphisms we looked for the same variants analysing 100 alleles at random. To achieve this goal we used, alternatively, restriction analysis and the minisequencing method with an automated capillary electrophoresis system and fluorescent-labelled dideoxynucleotides. Results and Conclusions: Two novel variants, one in each patient, were identified. One, involved codon 23 (TCA → TAA) and the other, codon 223 (CAA → TAA). Analysis of 50 DNA samples, randomly chosen, revealed that all were homozygous for the wild variant at codon 23. One of them was heterozygous for the variant CAA → TAA at codon 223. This sample was found to correspond to a Caucasian female in whom serum TBG proved to be not detected. Since both variants identified result in stop codons likely to induce truncated TBG proteins, they are probably responsible for the TBG phenotype observed in the individuals studied.