Objectives. Histomorphologic studies have provided evidence of prostate-specific antigen (PSA)-producing tissue in the female urethra. Some urine samples from women in a small series were positive for PSA, but no systematic investigation of this subject has been done to date. Methods. In a prospective study, we analyzed whether PSA occurs in the urine of women and what factors induce detectable PSA levels. The urine samples of 217 women were analyzed (Hybritech-Tandem E-PSA) under standardized conditions. The impact of urine pH and volume was investigated, and the results were correlated with clinical data (age, residual urine, urinary tract infection and prior sexual intercourse within 48 hours). Results. A positive PSA level greater than the detection limit of 0.1 ng/mL was found in 11% of the analyzed samples; their mean value was 0.29 ng/mL. pH correction did not result in a significant difference. The voiding volume had no influence on the PSA level. Among the cases of detectable PSA, women younger than 50 years of age (n = 14) had a mean PSA of 0.34 ng/mL and those older than 50 years (n = 9) a mean of 0.23 ng/mL. One of 9 women with and 22 of 208 women without residual urine volume had a detectable PSA level, as did 0 of 20 with and 23 of 197 women without urinary tract infection, and 3 of 7 with and 20 of 210 women without prior sexual intercourse within the previous 48 hours. None of the differences were significant. Conclusions. A urine PSA level was detected in 11% of all women studied, with PSA values apparently age dependent. Any urine portion is suitable for analysis. No influence was determined for residual urine volume or urinary tract infection. Sexual intercourse may cause detectable PSA values, but the data of this study did not provide sufficient evidence for this hypothesis.