Serum prostate-specific antigen (PSA) assays differ in calibration and response to different PSA forms. We examined intermethod differences in total PSA (tPSA) and free PSA (fPSA) measurements. We tested 157 samples with tPSA concentrations of 2 to 10 ng/mL (2-10 microg/L) using 6 PSA/fPSA method pairs and 1 tPSA method: ADVIA Centaur (complexed and total; Siemens Diagnostics, Tarrytown, NY), ARCHITECT i 2000(SR) (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), IMMULITE 2000 (Siemens Diagnostics), Modular E170 (Roche Diagnostics, Indianapolis, IN), UniCel DxI 800 (Beckman Coulter, Brea, CA), and VITROS ECi (tPSA only; Ortho-Clinical Diagnostics, Raritan, NJ). Regression analysis was performed for PSA, fPSA, and percentage of fPSA with the ARCHITECT i 2000(SR) comparison method. Differences between test and comparison methods were estimated at 2.5, 4.0, and 10.0 ng/mL (2.5, 4.0, and 10.0 microg/L) for tPSA and 15%, 20%, and 25% for percentage of fPSA. Relative differences were more than 10% at 4.0 ng/mL (4.0 microg/L) tPSA for the Centaur, IMMULITE, ECi, and DxI methods. At 20% fPSA, the relative difference was more than 10% for all methods except the AxSYM. Additional harmonization is needed for tPSA and fPSA methods.