We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar to a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Ifi-201, Ifi- 202, Ifi-203, etc, that are not regulated in a cell- or tissue-specific fashion. However, a new member of the Ifi-200 gene family, D3, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Ifi- 200 gene family, is also repeated in the recently characterized human IFI 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon gamma. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response element in the 5′ untranslated region, was significantly upregulated in human monocytes exposed to interferon alpha. Characterization of the MNDA gene showed that it is a single-copy gene and localized to human chromosome 1q 21–22 within the large linkage group conserved between mouse and human that contains the Ifi-200 gene family. The IFI 16 gene is also located on human chromosome 1q. Our observations are consistent with the proposal that the MNDA is a member of a cluster of related human interferon-regulated genes, similar to the mouse Ifi-200 gene family. In addition, one mouse gene in the Ifi-200 gene family and the human MNDA and IFI 16 genes show expression and/or regulation restricted to cells of the hematopoietic system, suggesting that these genes participate in blood cell-specific responses to interferons.