Objectives Clinical laboratories store filter paper samples used in neonatal screening for various periods of time after performing hormonal measurements. However, due to lack of data concerning specimen stability, it is unclear for how long these samples should be stored. The objective of this study is to determine the stability and reproducibility of thyroid-stimulating hormone (TSH), thyroxine (T 4 ) and 17-hydroxyprogesterone (17-OHP) measurements in filter paper blood samples stored for up to 60 months. Methods Two hundred and twenty-eight blood samples, drawn between the second and the fourth day of life, were divided into seven distinct groups and kept at 4-88C for one day or 2, 12, 24, 36, 48 or 60 months after basal hormonal measurements. In each group, TSH, T 4 and 17-OHP levels were initially assayed 24-48 hours after collection (basal) and repeated once at the end of storage timing. All the measurements were performed by time-resolved fluorometry (1235 AutoDELFIA, Wallac Oy, Turku, Finland). Repeated and basal levels of each hormone were compared within the same group by Student's paired t-test. Differences were considered significant at P , 0.05. Results Compared with basal measurements, TSH and T 4 levels declined significantly only when these hormones were re-assayed at 48 or 60 months of sample storage. In contrast, 17-OHP concentrations decreased earlier, starting at 24 months and continuing throughout the remaining period. Conclusion Our data suggest that neonatal screening of filter paper samples kept at 4 -88C are reliable for repeating the hormonal measurements when specimens are stored for up to one year, in the case of 17-OHP, or three years, in the case of T 4 and TSH.