The bacterial and archaeal diversity of deep groundwater systems was investigated based on 16S rRNA-SSCP (single strand conformation polymorphism) fingerprints. The study site included five boreholes along the projected Brenner Base Tunnel in the central Alps of Tyrol, Austria. To obtain representative samples, packer-sealed fractures were sampled at specific depths between 105 and 780 m below surface. Sequence analysis of SSCP bands obtained from 13 samples showed that between 29 and 62 % of the phylotypes belonged to a variety of Proteobacteria including representatives of typical freshwater bacteria of the genera Acidovorax, Aquabacterium, and Sphingomonas. Bacteroidetes (especially Flavobacterium), Firmicutes (Acetobacterium), and candidate division OP3-related sequences were observed in the majority of the analysed groundwaters. On average, 14 % of the detected prokaryotic phylotypes were affiliated with Archaea, comprising the phyla Euryarchaeota, Crenarchaeota and Thaumarchaeota. Most of the archaeal sequences showed low similarities to known cultivated species, with exception of two sequences having 98 % similarity to Methanosaeta sp. A considerable number of thaumarchaeal sequences belonged to two groups related to Nitrososphaera and Nitrosopumilus phylotypes. An environmental clustering of the groundwater samples, based on the bacterial and archaeal phylogeny, revealed a clear distribution pattern of the samples (sites and depths) reflecting the hydrochemical characteristics and underlying geologies.