Normal human skin is remarkably resistant to infection from the large numbers of microorganisms that routinely colonize its surface. In addition to the role of skin as a mechanical barrier, it has long been recognized that skin and other epithelia can produce a range of anti-microbial chemicals that play an important part in eliminating potential cutaneous pathogens. Anti-microbial peptides are an important evolutionarily conserved innate host defense mechanism in many organisms. Human β defensin-1 and -2 are cysteine-rich, cationic, low molecular weight anti-microbial peptides that have recently been shown to be expressed in epithelial tissues. In this study, we describe the characterization of human β defensin-1 and -2 mRNA and peptide expression in normal human skin. Using reverse transcription–polymerase chain reaction we demonstrate that human β defensin-1 is consistently expressed in skin samples from various body sites. Human β defensin-2 demonstrates expression that is more variable and is more readily detectable in facial skin and foreskin compared with skin from abdomen and breast. In situ hybridization localizes the human β defensin-1 and -2 transcripts to keratinocytes within interfollicular skin. Using specific antibodies, we have shown that human β defensin-1 and -2 peptides are localized to the Malpighian layer of the epidermis and/or stratum corneum and that there are interindividual and site-specific differences in intensity of immunostaining and the pattern of peptide localization. The localization of human β defensins to the outer layer of the skin is consistent with the hypothesis that human β defensins play an essential part in cutaneous innate immunity.