In the present study, the role of B-1 cells in hexachlorobenzene (HCB)-induced autoimmune aberrations in the Wistar rat was investigated. To that end, male and female rats were exposed to a semi-synthetic diet containing 0 or 1000 mg HCB/kg food for 3 weeks. After dissection, serum was prepared form coagulated blood to determine (auto)antibody levels, and spleens and lymph nodes were isolated and weighed. Cell suspensions were prepared, counted and analysed for B- and T-cell subsets by flow cytometry. Quantification of antibody-secreting cells (ASC) in spleen cell suspensions was done with an ELISPOT assay. Previous findings that HCB treatment induced an increase of relative lymph node and spleen weights and serum (auto)antibody levels were confirmed, while it appeared that numbers of some lymph nodal, and of the splenic large cell populations, were elevated as well. HCB treatment did not change subsets of lymph nodal T and B cells, but elevated the absolute numbers of large splenic CD4+ T cells by about 70%, IgMdull/IgDbright B cells by about 60%, and IgMbright/IgDdull B cells by about 200% cells of control numbers, and the absolute numbers of splenic IgM and IgG (auto) ASC by 300-400% of the control numbers. As splenic IgMbright/IgDdull numbers and ASC numbers correlated with statistical significance, the results indicate that HCB treatment selectively activates rat splenic B-1 cells, which may underlie the elevation of serum autoantibody levels.