Our objective was to evaluate performance of the clinical laboratories for the detection of antinuclear antibodies (ANA) by using indirect immunofluorescence method (IIF), in France. A national external quality assessment (EQA) on ANA detection was organized by the French health products safety agency once a year since 1998. Between 606 to 687 laboratories together with six university reference laboratories experienced in performing tests in autoimmunity participated in the six-year consecutive survey. Each laboratory had to answer to methodological procedures and give coded responses. Variability in IIF methodological procedure was observed. Use of inappropriate microscope magnifications for reading slides or nonconventional cutoff dilution of serum were pointed out to concerned laboratories. Concerning ANA measurement, the rate of good responses ranged from 92.7% to 99.5% of the laboratories when the samples contained ANA. A wide dispersion of ANA titers obtained on a same sample was repeatly observed every year. Misinterpretation of particular fluorescence pattern was noticed. On ANA negative sample, the rate of good responses was 94.3%. In conclusion, ANA detection in routine practice is far from being standardized. However, EQA may have an impact on ANA detection performance when it is conducted on several consecutive year surveys, by providing advice for participating laboratories to limit inter laboratory variations related to methodological procedures.