Autoimmunity is a state of permanent persistence of the autoantibodies and autoreactive T lymphocytes in patients’ plasma engaged in pathomechanism of the connective tissue diseases (CTDs). Specific autoantibodies are recognized as “markers” for particular types of diseases. Such antibodies are known as anti-nuclear antibodies (ANA) and are present in different percentages in CTD patients. However, in about 10-25% of tested sera from rheumatic patients, where the ANA test is positive, estimation of particular ANA specificities is unsuccessful. One of possible reasons for such seronegativity is complexemia, usually correlated with the acute phase of the disease. The goal of researches was the analysis of the CIC (PEG-6000 sediments) for the presence of “marker” autoantibodies in a group of these “trouble-making” sera. Researches cover a group of 588 sera originating from Clinics and Outpatient Clinic of Institute of Rheumatology. In the sera a titre and type of ANA pattern (IIF-method) and presence of selected auto-antibodies (done by ELISA and Western-blotting method) were estimated. In 69 (11.7%) ANA-positive (titre ≥ 1/160) sera, the autoantibody estimation gave a negative result. Then, we proposed a new methodological approach to the analysis of ANA-positive sera where classical methods of ANA specificity assessment was unsuccessful. The new approach is based on the analysis of the ANA specificity not in sera but in PEG-6000 sediments (or possibly in γ-fractions from PEG sediments), which are generally composed of different serum macroglobulins and also contain a concentrated circulating immune complexe (CIC) fraction. Using this new method it was possible to estimate autoantibody specificity in 84% of the sera formerly negative, where the standard methodology was useless. It is worth noting that in about 87% of PEG sediments more than one ANA specificity appears (Fig. 1). Among 133 antibody specificities found in 69 sera (on average, 2 different antibodies per serum), antibodies to chromatin antigens (anti-dsDNA, Hp) were positive in about 50% of all estimated autoantibodies, whereas antibodies to ENA-antigens were positive in 32% and in the case of residual autoantibodies, positivity was discovered only in 10.5% of all cases (Fig. 2). Additionally, in most (72%) PEG-6000 sediments tested for autoantibodies, the positive reaction (weakness of the lines on Western blots in relation to controls) after treatment (incubated with PEG sediment blots) with anti-human F(ab)2-IgG antiserum and in 30% after incubation with anti-h-Fc-IgG antiserum was observed. The effect of weakening of lines on the Western blots after incubation with anti-F(ab)2 and anti-Fc sera was similar, independently of ANA antibody specificity, what shows that autoantibodies are located on the surface of the immune complexes (Fig. 3, Table I). One can hope that a new methodological approach developed after necessary adjustment (simplification) needed for routine antibody diagnostics, will be useful in diagnosis of marker autoanti­bodies not only in connective tissue diseases.