The human IFI 16 gene encodes a nuclear protein whose expression is associated with activation and differentiation of cells of the myeloid lineage, and which has recently been postulated to be a frequent target of human autoantibodies. We have used a unique monoclonal antibody in immunofluorescence studies to define the intranuclear distribution of IFI 16 antigen in primary human leukocyte sub-populations. We demonstrate that IFI 16 is expressed both within the nucleoli and nucleoplasm of mononuclear cells, but is not present in granulocytes. IFI 16 expression correlated with that of the known nucleolar antigen B23, suggesting that the function of IFI 16 is restricted to cells that have nucleoli and therefore are not terminally differentiated. These findings were supported by immunofluorescence studies in IFN-gamma-stimulated HL-60 cells and by biochemical fractionation of Daudi nuclear extract. IFI 16 was extracted from whole nuclei at approximately 200-300 mM NaCl, within the range described for known transcription factors. Together with our previous findings, these results support a possible role of IFI 16 in binding nucleic acid in vivo. Furthermore, a DNA-binding site was localized within a fusion protein containing the amino-terminal 159 amino acids. As the nucleic acid-binding domains of several proteins are targeted by autoantibodies in SLE, this region may be of significance in the pathogenesis of autoimmune disease.