In order to identify novel proteins produced by activated macrophages, a cDNA library was made from cultures of the mouse macrophage-like cell line RAW 264.7 that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the library was screened by differential plaque hybridization. A cDNA clone was isolated that detected a 1.4-kilobase mRNA that accumulated dramatically in response to the spleen cell conditioned medium. The 1.4-kilobase mRNA encodes a predicted protein of 98 amino acids, designated CRG-2, molecular weight (Mr) 10,781, with a 21-residue signal peptide. The amino acid sequence indicates that CRG-2 is a member of the platelet factor 4 family (PF4) of cytokines. The CRG-2 mRNA was induced by alpha-, beta-, and gamma-interferons (IFNs) and by lipopolysaccharide. In response to IFN-gamma, the CRG-2 mRNA level reached a peak between 3 and 6 h. The accumulation of CRG-2 mRNA was not blocked by cycloheximide. Among the known members of the PF4 family, CRG-2 is most closely related to the interferon-inducible human protein IP-10. The 5'-flanking region of the crg-2 gene was isolated, and comparisons between crg-2 and IP-10 genes, mRNAs, and proteins reveal conserved features of possible functional importance. CRG-2 may play a role in host defense, particularly in the response to viral infection.