Necrotizing hepatopancreatitis (NHP) is a disease of farm-raised Pacific white shrimp (Penaeus vannamei) caused by a pleomorphic intracellular bacterium. A DNA probe that is specific for the etiologic agent of necrotizing hepatopancreatitis was devised and tested in an in situ hybridization assay. A procedure was developed for labeling a single-stranded DNA probe with digoxigenin by the polymerase chain reaction. The DNA probe encompasses the V1 and V2 variable regions of the 16S ribosomal RNA (rRNA) gene and is designed to hybridize to complementary sequences of the 16S rRNA of the NHP bacterium. The probe was tested on fixed, paraffin-embedded specimens, and an intense, specific hybridization signal was localized to the cytoplasm of hepatopancreatic epithelial cells that were infected with the NHP bacterium, as demonstrated by serial sections stained with hematoxylin and eosin or the Steiner and Steiner method. Negative results were obtained from normal shrimp and from shrimp infected with Vibrio spp. The specificity of the probe was confirmed using either mammalian or avian tissues infected with other intracellular bacteria, including Ehrlichia canis, Salmonella enteritidis, Brucella abortus, and Chlymidia spp., and using another species of shrimp (P. monodon) infected with a different rickettisa-like intracellular bacterium.