The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bane marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL- 60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A(-/-) bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A(-/-) macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A(-/-) mice showed no increased mortality or bacterial burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.