Anti-nuclear antibodies (ANA), as detected by indirect immuno-fluorescence, are hallmarks of autoimmune connective tissue diseases. Identification of the specificity for extractable nuclear antigens (ENA) is warranted because this may further differentiate between the distinct types of autoimmune connective tissue diseases. In recent years several different ENA, as recognized by ANA, have been identified and the knowledge of the molecular structure has been expanded. Together with technical developments this has enabled the introduction of several new anti-ENA antibody detection systems. In this review we will discuss the main logistic aspects of anti-ENA antibody testing that have to be solved in order to come to a consensus in order to deal with new developments in this field. We conclude that: 1. a positive ANA test should, depending on the titre and pattern, be followed by an anti-ENA antibody assay, 2: to fully appreciate the value of the new anti-ENA antibody detection systems a large, multicenter clinical evaluation is required, and 3: proper interpretation of reported test results requires that the clinician is aware of the way anti-ENA antibodies are detected and reported.