We have developed a method to enrich, isolate, and identify Thiothrix spp. in environmental samples. This procedure employs low concentrations of organic compounds, the addition of reduced sulfur compounds (sulfide or thiosulfate), and preparation with spring water that contains Thiothrix spp. The enrichment enhanced identification of Thiothrix spp. by promoting deposition of intracellular sulfur granules and inhibiting overgrowth by other bacteria. The relatively high calcium content of the spring water contributed to the culture procedure. With this technique, Thiothrix spp. were observed in two activated sludge systems, a municipal water storage tank, three springs, and four underground freshwater caves in the phreatic zone of the Floridan aquifer. Two different Thiothrix cultures have been isolated from a freshwater cave and a water storage tank by this procedure. It appears that media prepared with spring water known to support Thiothrix spp. can be designed to provide highly selective methods for isolation of Thiothrix spp. from a wide range of environments.