In the Lusatia area a pilot plant for the treatment of acidic mine waters by microbial iron oxidation and a concomitant iron hydroxysulfate precipitation is operated. Molecular based studies of acidic waters from this iron hydroxysulfate producing pilot plant revealed the presence of 16S rRNA gene sequences from undescribed iron-oxidizing bacteria. Most of these were related to autotrophic Betaproteobacteria (see Heinzel et al. IBS 2007, poster number 98). For cultivating different iron-oxidizing bacteria water samples from this pilot plant were directly plated on various solid media. Double-layer plates were used, with a heterotrophic Acidiphiliumstrain in the underlayer, because of the high sensitivity of autotrophic bacteria towards organic substances. The media contained different iron-substrates with and without organic carbon sources. Colonies appeared at the latest after three weeks and they were encrusted with ferric iron. The phylogeny of the isolated strains was determined and the physiological requirements, like temperature, pH optima, preferred carbon source and iron concentrations, were analyzed. Many isolates which were related to Acidithiobacillus ferrooxidans strains could be cultivated, as well as an isolate related to the genus Thiomonas and one isolate related to a Ferribacter polymyxa species. The Thiomonas-like isolate showed best growth in media containing tryptone soya broth, sodium thiosulfate and ferrous sulfate at pH 2.5 and 30°C. The other Betaproteobacterium grew on ferrous sulfate medium at pH 2.5 between 16 and 37°C. In liquid culture experiments the cells of both isolates were attached to the iron minerals built in the medium. These physiological characteristics of the isolates helped to vary parameters in the pilot plant to optimize the process of iron oxidation and improved waste water remediation.