Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. Mononuclear cells from cord blood samples of different newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. Six different, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing lambda clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. The secreted Abs did not affect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing.