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A molecular characterization of pristine and petroleum hydrocarbon-contaminated Alpine soils sampled in Tyrol (Austria) was performed. To identify predominant bacteria, PCR-amplified 16S rRNA gene fragments from five pristine and nine contaminated soils were analysed using denaturing gradient gel electrophoresis (DGGE). Sequencing and phylogenetic analyses demonstrated that the majority of the DGGE bands represented bacteria in the Actinobacteria and Proteobacteria phyla: 18 and 73%, respectively, in pristine soils, compared with 20 and 76%, respectively, in contaminated soils. A different distribution pattern of bacterial classes in the Proteobacteria was observed between pristine and contaminated soils. The relative proportion of microorganisms belonging to the Alphaproteobacteria was larger in pristine (46%) than in contaminated (24%) soils, while Betaproteobacteria and Gammaproteobacteria were detected only in the hydrocarbon-contaminated soils. This result compared favourably with earlier work in which hydrocarbon-degradation genotypes, largely pseudomonads and Acinetobacter, belonging to the Gammaproteobacteria, were enriched following oil hydrocarbon contamination. In contrast, members of the Actinobacteria phylum, represented by Rhodococcus and Mycobacterium, were found in pristine soils where contamination events had not occurred. The results demonstrate a significant shift in the microbial community structure in Alpine soils following contamination. Furthermore, more potentially novel phylotypes were found in the pristine soils than in the contaminated soils.