The aim of this work was to study possible differences in the humoral response against autologous and heterologous (bacterial and mammalian) ssDNA in children with diffuse connective tissue diseases (DCTD) compared with age matched controls. We found that IgM anti ssDNA were significantly increased in systemic lupus eritematosus (SLE) and in juvenile arthritis (JA), but not in juvenile dermatomyositis (JDM). IgG anti ssDNA were significantly elevated only in children with SLE. We next evaluated the binding specificity to human and bacterial ssDNA by inhibition assays. We found that SLE and JA sera recognised epitopes shared in common to endogenous and bacterial ssDNA. In contrast, in normal subjects IgG binding to bacterial DNA was not inhibited by human DNA, while IgG anti human ssDNA were cross reactive with the bacterial antigen. These data suggest that natural antibodies (IgM) producing cells are activated in some but not all DCTD, and that normal children have different reactivity against autologous and heterologous ssDNA with respect to SLE and JA patients.