Differences in stability of the free and complexed molecular forms of prostate-specific antigen (PSA) may influence the clinical utility of assays for these forms, as well as the calculated ratios to total PSA (tPSA), such as percent free PSA (fPSA) and percent complexed PSA (cPSA). The objective of this study was to directly compare the short-term stability of fPSA and cPSA under different storage conditions. Specimens (3 with prostate cancer, 3 biopsy-negative without cancer, 2 normal) from 8 men were analyzed at baseline within 2 hours of collection, and at 4 hours, 8 hours, 24 hours, 48 hours, and 1 week after storage at room temperature, 4 degrees C, or -20 degrees C. Serum specimens were analyzed in duplicate on the Bayer Immuno 1 analyzer (tPSA, cPSA) and on the Beckman Coulter Access analyzer (tPSA, fPSA Tandem assays). Baseline tPSA values ranged from 0.7 to 62.0 ng/mL, with a median of 7.9 ng/mL (Immuno 1). Overall, all forms of PSA were stable up to 24 hours at the 3 temperatures, with the exception of fPSA and percent fPSA, which decreased when stored at 4 degrees C. After 1 week, tPSA levels decreased when stored at room temperature and at 4 degrees C, as did cPSA stored at room temperature. Over the 7 days, percent cPSA was stable at room temperature, but increased at 4 degrees C. There were no significant changes in any PSA form or calculated ratio with storage at -20 degrees C for up to 1 week. In summary, in the short term (<1 week), fPSA is less stable with storage than tPSA or cPSA in a time- and temperature-dependent fashion. Thus, specimen handling should be considered when interpreting PSA results. It is recommended that specimens not analyzed the same day (within 8 hours of collection) be stored frozen at -20 degrees C.