Antinuclear antibodies (ANAs) are regarded as a hallmark of connective tissue diseases (CTDs) and play a key role in their diagnosis, but the value of some particular antibodies in management of patients and the disease prognosis is controversial. The mechanism underlying the production of ANAs in CTDs, other chronic inflammatory conditions and even in healthy people, is not completely elucidated. Anti-DFS70 antibodies connected with the dense fine speckled autoantigen of 70 kD, known as the lens epithelium-derived growth factor p75, are a subgroup of ANAs. Their presence and coexistence with other antibodies and their clinical significance are the matter of debate. Based on literature data, the authors focused on current knowledge explaining the role of anti-DFS70 antibodies in selected CTDs. However, the literature data is ambiguous and does not fully support the validity of the anti-DFS70 assay for a specific CTD diagnosis. Most researchers claim that the presence of anti-DFS70 as the only one usually exclude the diagnosis of CTD. Nevertheless, its coexistence with other ANAs is not an excluding factor but has predictive value due to more favorable course of CTD. Such situations may also suggest an enhanced risk of the development of a CTD in the future. Although more studies are needed in this field, it seems reasonable to ascertain the presence of anti-DFS70 in routine clinical practice.