Bacterial strains A2 and HE5, isolated by enrichment culture techniques, were shown to produce extracellular, nondialyzable materials which disperse limestone powders in water. These materials are referred to as biodispersans. Strains A2 and HE5 were classified as Acinetobacter calcoaceticus by physiological and genetic tests. An assay for limestone-dispersing activity was developed which is based on the settling time of a standard 10% limestone-in-water mixture. The assay was proportional to biodispersan concentration between 40 and 100 mug/ml. Dispersion was optimum between pH 9 and 12. Phosphate (2 mM) and magnesium (8 mM) ions caused a 50% inhibition of activity. An optimized medium for the production of biodispersan was developed with ethanol as the substrate. Biodispersan was produced only after the bacteria completed their exponential growth phase and continued during the stationary phase, reaching over 4 g of crude biodispersan per liter.