Objective. Peripheral blood cells (PBMCs) from some patients with systemic sclerosis (SSc) express an interferon-α (IFNα) signature. The aim of this study was to determine whether SSc patient sera could induce IFNα and whether IFNα induction was associated with specific autoantibodies and/or clinical features of the disease. Methods. SSc sera containing autoantibodies against either topoisomerase I (anti-topo I; n = 12), nucleolar protein (ANoA; n = 12), or centromeric protein (ACA; n = 13) were cultured with a HeLa nuclear extract and normal PBMCs. In some experiments, different cell extracts or inhibitors of plasmacytoid dendritic cell (DC) activation, Fcγ receptor II (FcγRII), endocytosis, or nucleases were used. IFNα was measured by enzyme-linked immunosorbent assay. Results. Topo I-containing sera induced significantly higher levels of IFNα as compared with all other groups. IFNα induction was inhibited by anti-blood dendritic cell antigen 2 (90%), anti-CD32 (76%), bafilomycin (99%), and RNase (82%). In contrast, ACAs induced low levels of IFNα even when necrotic, apoptotic, or demethylated extracts were used, despite the fact that CENP-B-binding oligonucleotide containing 2 CpG motifs effectively stimulated IFNα. IFNα production was significantly higher in patients with diffuse SSc (mean ± SEM 641 ± 174 pg/ml) than in those with limited SSc (215 ± 66 pg/ml) as well as in patients with lung fibrosis than in those without. Conclusion. Autoantibody subsets in SSc sera differentially induce IFNα and may explain the IFNα signature observed in SSc. IFNα is induced by plasmacytoid DCs and required uptake of immune complexes through FcγRII, endosomal transport, and the presence of RNA, presumably for interaction with Toll-like receptor 7. The higher IFNα induction in sera from patients with diffuse SSc than in those with limited SSc as well as in sera from patients with lung fibrosis suggests that IFNα may contribute to tissue injury.