Quantitative real-time PCR techniques are increasingly being used for the measurement of nucleic acids in research applications as well as in the clinical laboratory. It is therefore important that external quality control programs (EQA) are implemented for the evaluation of the analytical aspects common to molecular tests based on quantitative PCR. The aim of this study was the development of an Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Man™ probes to compare the analytical performance of 42 laboratories. Participants were provided with a set of reagents (cDNA for reference curve preparation, primers-probe mix and three unknown samples) and requested to perform a conventional assay using the master mix employed in their laboratories. The quantitative results in unknown samples were analyzed. The results of our study showed clear heterogeneity in performance. Two of the 42 laboratories provided results indicating contamination during the experiment, whereas six did not provide values for at least one of the six standard points. Only 12 laboratories gave results that were both precise and accurate for all the samples tested. Regarding imprecision, 17 laboratories appeared to deviate in at least one result, whereas inaccuracy showed an inverse dose-dependent trend. Finally, 12 laboratories were not able to measure the sample with the lowest concentration. Ten of these laboratories were equipped with the same instruments. The results of this first round of analytical EQA of real-time PCR-based methods seem to indicate high variability among laboratories carrying out the same experimental protocol. These findings could have implications for any assay based on this type of technique. This survey demonstrates the importance of experimental EQAs of methodological proficiency testing. Our approach has proved useful for comparing the analytical aspects shared by all diagnostic laboratories applying quantitative assays for the measurement of nucleic acids based on the use of Taq-Man™ probes and real-time platforms.