To check the efficiency and the sensitivity of the polymerase chain reaction, it is advisable to apply standard molecules as indicators of the procedures. It is important to detect false negative results depending on pipetting errors or inhibition of the amplification, often observed when PCR is performed with complex samples, such as faeces and tissues. We have developed internal control molecules, termed mimics, to be used in two diagnostic PCR systems for the detection of the M. avium subsp. paratuberculosis specific element IS900 . Two oligonucleotides with the IS900 PCR primers p36 and p11 as 5'overhang and two oligonucleotides with the IS900 nested PCR primers s204/s347 and s749/s535 as 5'overhang, were constructed to amplify a region of 387 bp and 635 bp, respectively, of a segment of the human actin gene inserted into the pUC18 plasmid. The resulting mimics had the target sequences for the IS900 PCR primers at both ends. The mimic amplicons were cloned in PCR- Amp SK(+) cloning vector and the purified plasmid was used in the PCR . The amount of mimic that could be used in the PCR without affecting the amplification of the segment of IS900, was determined to be 103-104 molecules in single PCR and 10 molecules in nested PCR. The use of mimics in PCR was evaluated with spiked samples and clinical samples which were previously found positive in culture. When M. avium subsp. paratuberculosis DNA was present at high concentration in a sample, the IS900 PCR product was detected, but not the mimic, due to competition. We did not observe any competitive effects on the IS900 amplification when the mimic was included in the PCR. The identical primer-binding nucleotide sequences allowed co-amplification of the IS900 element and the mimic in the same tube, and simultaneously, the size difference allowed easy discrimination between their PCR products. Using the mimic undoubtedly facilitated the interpretation of negative PCR results and it was easy to identify samples which were inhibiting the amplification.