Mammoth Cave, a large limestone cavern in Mammoth Cave National Park in the Central Kentucky karst, was first mined for saltpetre in 1808 and was a major source of nitrates used in the manufacture of gunpowder during the War of 1812. The mechanism of saltpetre formation is unknown, although hypotheses encompassing both biotic and abiotic functions have been suggested. Present studies were conducted in various saltpetre caves using species specific fluorescent antibodies in order to determine if the chemoautotroph, Nitrobacter, were present. Population densities and species distribution of Nitrobacter were studied in relation to chemical and physical parameters for over 200 sediment samples from Mammoth Cave. Both the isolation and immunofluorescence data indicate that Nitrobacter are present in relatively high population densities in Mammoth Cave sediments, and that such bacteria are common among saltpetre caves in the southeastern United States. Immunofluorescence data further indicates that N. agilis dominates the Nitrobacter population in Mammoth Cave. The possibility that Nitrobacter is the etiological agent for saltpetre formation is suggested.