The stability of total (t) and free (f) prostate-specific antigen (PSA) in male serum specimens stored at -70 degrees C or lower temperature for 4.7 to 4.9 years was studied. Until now, the stability of these analytes in serum has not been evaluated systematically beyond 2 years of storage at -70 degrees C. Aliquots of frozen serum were thawed in 2001 and 2006 and assayed for tPSA and fPSA using a Dade Behring Dimension(R) RxL analyzer and reagents. tPSA values ranged from 0.07 to 69.94 and 0.00 to 69.83 ng/mL in 2001 and 2006, respectively, whereas fPSA values for the tested specimens ranged from 0.02 to 5.72 and 0.00 to 5.92, respectively. Deming regression analyses showed agreement in assay values over time as tPSA values yielded a slope of 1.0112 and a y-intercept of 0.0195; fPSA values produced a slope 1.0538 and a y-intercept of -0.0442; f/tPSA values yielded a slope of 0.9631 and a y-intercept of 0.1195. A Bland-Altman analysis of the data demonstrated analyte and ratio stability over this time period. We conclude that serum, when collected properly and stored at -70 degrees C or lower temperature, may be used for tPSA and fPSA clinical studies for at least 5 years after collection.