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Juvenile idiopathic arthritis (JIA) encompasses a heterogeneous group of diseases. The appearance of antinuclear antibodies (ANAs) in almost half of the patients suggests B cell dysregulation as a distinct pathomechanism in these patients. Additionally, ANAs were considered potential biomarkers encompassing a clinically homogenous subgroup of JIA patients. However, in ANA+ JIA patients, the site of dysregulated B cell activation as well as the B cell subsets involved in this process is still unknown. Hence, in this cross-sectional study, we aimed in an explorative approach at characterizing potential divergences in B cell differentiation in ANA+ JIA patients by assessing the distribution of peripheral blood (PB) and synovial fluid (SF) B cell subpopulations using flow cytometry. The frequency of transitional as well as switched-memory B cells was higher in PB of JIA patients than in healthy controls. There were no differences in the distribution of B cell subsets between ANA- and ANA+ patients in PB. However, the composition of SF B cells was different between ANA- and ANA+ patients with increased frequencies of CD21\(^{lo/−}\)CD27\(^−\)IgM\(^−\) “double negative” (DN) B cells in the latter. DN B cells might be a characteristic subset expanding in the joints of ANA+ JIA patients and are potentially involved in the antinuclear immune response in these patients. The results of our explorative study might foster further research dissecting the pathogenesis of ANA+ JIA patients.