Quantification of DNA in real-time using the Light Cycler is increasingly being used for the detection and follow-up of various infectious and other diseases. We evaluated the effect of two parameters, namely the presence of nonspecific DNA and prior repeated freezing and thawing on the accurate quantification of DNA extracts from the RH strain of Toxoplasma gondii by the SYBR Green I and the Hybridization Probe techniques. For both parameters, a high copy number sample containing 5x10(5) parasites/extract and a low copy number sample containing 100 parasites/extract were tested. Reliable quantification was possible in the presence of up to 200 ng of nonspecific DNA by the SYBR Green I technique and up to 1000 ng by the Hybridization Probe technique as compared to the company threshold of 50 and 500 ng, respectively. As tissue samples usually contain more than 200 ng of nonspecific DNA, the ideal choice is the Hybridization Probe technique. The stability of DNA extracts after repeated freeze-thaw cycles was found to be dependent on the volume in which they were stored. Samples stored in 100-microl total volumes were not stable after 3 freeze-thaw cycles, whereas those stored in 1-ml total volumes were stable after 14 freeze-thaw cycles.