Reliable reference ranges are important in the interpretation of laboratory data, and it is incumbent on each laboratory to verify that the ranges they use are appropriate for the patient population they serve. The objective of this study was to determine population-specific reference ranges for thyroid stimulating hormone (TSH), free thyroxine (fT4), free triiodothyronine (fT3) and total triiodothyronine (TT3) on the Abbott ARCHITECT i2000 analyzer. For this study, we used human serum samples collected from a population in Castilla y León, Spain. Serum samples were collected from 304 individuals (male, n=151; female, n=153; age 12–94 years) representing outpatients (n=100), hospitalized patients (n=104) and apparently healthy subjects (n=100). Individuals taking any medications, with a history of thyroid disorder, or severe non-thyroidal illness were excluded from the study. For healthy subjects, the following reference intervals were determined: TSH, 0.51–5.95 mIU/l; fT4, 0.84–1.42 ng/dl (10.77–18.21 pmol/l); fT3, 1.48–3.37 pg/ml (2.27–5.18 pmol/l); and TT3, 0.65–1.46 ng/ml (1.00–2.24 nmol/l). In this group, TSH and fT4 showed significant differences between men and women, but fT3 and TT3 did not. Conversely, fT3 and TT3 showed significant age-related differences, but TSH and fT4 did not. Within the outpatient group, no significant differences were seen between men and women for any of the hormones, but age-related differences were significant for fT3 and TT3. Within the hospitalized patient group, significant differences between men and women were found for TSH only, and age-related differences were significant for TSH, fT3 and TT3. Our findings are basically in accordance with previously published results for fT3, TT3 and TSH, but for fT4 our results differ from other data in the literature. This highlights the need for laboratories to confirm that the reference ranges they use are appropriate for the population they serve.