OBJECTIVES: In our study, we evaluated the antinuclear antibodies (ANA) staining patterns and titres in juvenile idiopathic arthritis (JIA) patients. METHODS: JIA patients were retrospectively assessed. ANA was identified by using indirect immunofluorescence assay on HEp-2 cells, with a positivity threshold set at a titre of >/=1/100. RESULTS: Eight hundred-five patients were evaluated [oligoarticular JIA (n = 396), enthesitis-related arthritis (n = 195), polyarticular JIA (n = 132), systemic JIA (n = 53), psoriatic arthritis (n = 20), and unclassified JIA (n = 9)]. The most frequently observed ANA nuclear staining patterns were AC-4/5 (fine or large speckled) in 29.7% of patients and AC-1 (homogeneous) in 25.9%. The most common ANA cytoplasmic staining pattern was AC-19 (dense, fine speckled) (17.6%). Most systemic and unclassified JIA patients were ANA negative. The most frequently detected ANA titre in oligoarticular JIA and enthesitis-related arthritis patients was 1/160 (P = .026 and P = .018, respectively), while in psoriatic arthritis patients, it was 1/320 (P < .001). In addition, uveitis and inflammatory bowel disease were most frequently associated with AC-4/5 & AC-19 patterns and an ANA titre of 1/160 (all P < .001). CONCLUSIONS: Our study showed that many JIA subtypes and JIA-related comorbidities were associated with the AC-4/5, AC-1, and AC-19 ANA patterns. However, multicentre studies in larger cohorts are needed to generalize these results.