Subterranean environments are potential sources for the isolation of novel microorganisms. Water and soil samples were collected at depths ranging from 10 to 1800 meters below the surface, and screening was carried out with aerobic rich and anaerobic minimal media. Two psychrotrophic anal three chemoautotrophic strains were isolated. One of the psychrotrophic isolates, designated SN16A, grew at temperatures between -5 and 37 degrees C with optimal growth between 25 and 30 degrees C. The other psychrotroph, designated KB700A, grew between -10 and 30 degrees C. Little difference in growth rate could be observed between 20 and 30 degrees C; however, this strain did not grow at 37 degrees C. KB700A utilized CO2 chemoautotrophically at 30 degrees C, using hydrogen as an energy source. Both strains were characterized biochemically. The complete 16S rRNA sequence of KB700A was 98.7% homologous with that of Pseudomonas marginalis. However, the 16S rRNA of SN16A showed only 95.4% identity at maximum-with the corresponding gene of Arthrobacter globiformis-suggesting that this strain may belong to a novel genus. Both strains exhibited the ability to produce hydrolytic enzymes on plate assays. Our results suggest that subterranean environments are promising sources for the isolation of psychrotrophic microorganisms.