Determination of prostate-specific antigen (PSA) is an established tool in detecting prostate cancer. However, the effect of physical activity on the PSA concentration in serum is controversial. We measured serum concentrations of PSA and prostatic acid phosphatase (PAP) in 301 healthy outpatients before and after they performed standardized exercise. Immediately after 15 min of exercise on a bicycle ergometer, their serum PSA concentrations increased by as much as threefold. The increase was age dependent and correlated to the PSA concentration before exercise. This increase was evident in both the free and complexed fractions of PSA. The amount of PSA secreted into blood depends on the volume of the prostate, whereas productivity of the prostate epithelium remains constant or increases slightly with age. We present cutoff values for clinical use. PAP was also increased, but to a lesser extent. The PSA and PAP secretion mechanisms differ. Our data suggest that extensive physical activity should be avoided before blood sampling for diagnostic purposes and, in case of an increase, the PSA concentration should be controlled after an exercise test.