A quantitative and systematic analysis is provided for ubiquitously present template DNA interfering with the quantification of human DNA by PCR. Two sources contributing to DNA background were identified. The first one is interpreted as DNA present in chemicals and on equipment and the second as caused by operator handling. The amounts were equivalent to 2.5 and 8.9 pg per mL of sample, and the estimated frequencies of contamination were 65 and 35%, respectively, resulting in an effective limit of detection of 17.4 pg/mL. Below this level--named effective laboratory background--a result could not be considered as authentic. Knowledge of these parameters is important for laboratories that analyze minute amounts of human DNA by PCR for purposes such as quantification, typing, and sequencing.