The secondary intracellular target of human neutrophil peptide-1 has been examined in M. tuberculosis H37Ra. Binding studies with radioiodinated HNP-1 revealed biphasic equilibrium binding kinetics with respect to time. The major site of HNP-1 binding was found to be plasma membrane/cell wall whereas the cytosol appears to be a secondary site. Among the different macromolecules examined, maximum inhibition (75%) was observed in DNA biosynthesis during treatment with HNP-1. The interaction of HNP-1 with mycobacterial genomic DNA on the basis of gel retardation assay revealed HNP-1 binding to DNA. These results indicate that HNP-1 has DNA as the secondary intracellular target for antibacterial action against mycobacteria.