Hepcidin is a urinary antimicrobial peptide synthesized in the liver. It was identified from human urine as a cysteine-rich peptide with three forms differing by amino-terminal truncation. Named for its liver origin and antimicrobial properties, the two predominant forms, Hepc20 and Hepc25, contain 20 and 25 amino acids, respectively, with all 8 cysteines connected by intramolecular disulfide bonds. Homologous liver cDNAs were found in species from fish to human, and a corresponding human genomic sequence was identified on chromosome 19. The full cDNA was 0.4 kilobase pairs, consistent with hepcidin mRNA size on Northern blots. The liver was the primary site of mRNA expression, and the encoded prepropeptide contains 84 amino acids, but only the 20–25-amino acid processed forms were found in urine. Hepcidin exhibits antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger, and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. It may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects. Innate immunity relies on various effector mechanisms, including disulfide-linked cationic antimicrobial peptides found in both plant and animal kingdoms. These peptides exhibit broad activity against bacteria, fungi, protozoa, and enveloped viruses. The vertebrate liver is centrally involved in innate immune response to infection. The "acute phase" response to infection or inflammation involves increased hepatic synthesis of secreted proteins involved in host defense. In contrast to the abundant fat body-derived antimicrobial peptides of insects, no vertebrate antimicrobial peptides originating in the liver have been described. This study reports the discovery of a novel hepatic antimicrobial peptide, hepcidin, whose processed form is found in urine. Hepcidin was purified from urine using cation exchange chromatography and RP-HPLC. Amino acid sequencing and MALDI-TOF-MS confirmed its identity. CD spectroscopy showed a structure with β-turns, loops, and distorted β-sheets. Northern blot analysis showed high expression in fetal and adult liver. cDNA cloning identified a homologue in various species, including pig, rat, mouse, flounder, and the long-jawed mudsucker. The gene consists of three exons and two introns, with the third exon encoding the peptides found in urine. Antimicrobial assays showed that hepcidin is active against various microbial strains, including bacteria and fungi. Germination assays demonstrated that Hepc20 was more potent than Hepc25 against Aspergillus species. FungicHepcidin is a urinary antimicrobial peptide synthesized in the liver. It was identified from human urine as a cysteine-rich peptide with three forms differing by amino-terminal truncation. Named for its liver origin and antimicrobial properties, the two predominant forms, Hepc20 and Hepc25, contain 20 and 25 amino acids, respectively, with all 8 cysteines connected by intramolecular disulfide bonds. Homologous liver cDNAs were found in species from fish to human, and a corresponding human genomic sequence was identified on chromosome 19. The full cDNA was 0.4 kilobase pairs, consistent with hepcidin mRNA size on Northern blots. The liver was the primary site of mRNA expression, and the encoded prepropeptide contains 84 amino acids, but only the 20–25-amino acid processed forms were found in urine. Hepcidin exhibits antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger, and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. It may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects. Innate immunity relies on various effector mechanisms, including disulfide-linked cationic antimicrobial peptides found in both plant and animal kingdoms. These peptides exhibit broad activity against bacteria, fungi, protozoa, and enveloped viruses. The vertebrate liver is centrally involved in innate immune response to infection. The "acute phase" response to infection or inflammation involves increased hepatic synthesis of secreted proteins involved in host defense. In contrast to the abundant fat body-derived antimicrobial peptides of insects, no vertebrate antimicrobial peptides originating in the liver have been described. This study reports the discovery of a novel hepatic antimicrobial peptide, hepcidin, whose processed form is found in urine. Hepcidin was purified from urine using cation exchange chromatography and RP-HPLC. Amino acid sequencing and MALDI-TOF-MS confirmed its identity. CD spectroscopy showed a structure with β-turns, loops, and distorted β-sheets. Northern blot analysis showed high expression in fetal and adult liver. cDNA cloning identified a homologue in various species, including pig, rat, mouse, flounder, and the long-jawed mudsucker. The gene consists of three exons and two introns, with the third exon encoding the peptides found in urine. Antimicrobial assays showed that hepcidin is active against various microbial strains, including bacteria and fungi. Germination assays demonstrated that Hepc20 was more potent than Hepc25 against Aspergillus species. Fungic