Environmental DNA extracted from cave sediments contains many DNA sequences, some of which encode bacterial 16S rDNA from the cave bacterial community. Prior efforts in our laboratory have led to the creation of a database of 16S rDNA sequences from a clone library of bacterial DNA extracted from cave sediments or bacteria cultured from cave sediments. Much of this work is focusing on a series of different environments in the Mammoth Cave System, including sediments within varying sized streams and vertical shafts. Cloned or cultured 16S rDNA sequences are aligned and compared with online software tools. Also in the database for each entry is an experimentally measured fluorescent fragment length that serves as a convenient biomarker for that particular bacterial 16S rRNA gene. Mixed bacterial 16S rDNA is amplified by polymerase chain reaction (PCR) and labeled with fluorescent dyes, followed by restriction digestion to generate a profile with many fragments representing the bacterial community diversity. The profile is interpreted with the aid of phylogenetic, DNA sequence, and biomarker information afforded by the cave bacterial 16S rDNA database and comprehensive online genetic depositories. Highly specific biomarkers have been developed to monitor two clones we have identified from Mammoth Cave and from isolated cave microenvironments elsewhere in Kentucky and in Alabama. We hypothesize that these organisms represent previously undescribed chemoautotrophic nitrite oxidizers that utilize carbonate minerals for growth in the virtual absence of organic carbon, thus contributing to limestone dissolution, cavern enlargement, sinkhole collapse, and karst aquifer evolution. We have also produced very different bacterial DNA profiles from sediments collected in caves of Sequoia/King's Canyon and Wind Cave National Parks. In collaboration with the National Cave and Karst Research Institute, the techniques described here will be used to monitor selected cave sediments in national parks across the USA.