Antibodies in the sera of patients with human granulocytic ehrlichiosis (HGE) commonly recognize a 44-kDa antigen. We cloned the gene encoding the 44-kDa protein of the agent of HGE (aoHGE) by probing an aoHGE lambda ZAP II genomic DNA expression library with sera from aoHGE-infected mice. The gene, hge-44, is part of a multigene family, with sequence similarity to the Anaplasma marginale msp-2 genes. RNA-PCR studies confirmed that hge-44 is expressed by aoHGE cultured in HL-60 cells and by aoHGE during murine infection. Recombinant HGE-44, expressed and purified as a glutathione transferase fusion protein, was used as the substrate in immunoblots to help diagnose HGE. Antibodies in eight sera from eight patients with HGE and in two sera from two aoHGE-infected mice bound recombinant HGE-44. Antibodies in the sera of healthy individuals or patients with Ehrlichia chaffeensis or Borrelia burgdorferi infection did not recognize HGE-44. We conclude that hge-44 is a member of a multigene family and that hge-44 is expressed and elicits specific antibodies during infection.