Temporal and spatial variation of soil bacterial communities was evaluated with both molecular and metabolic profiling techniques. Soil cores (20 cm deep) were taken from an upland grassland in the Scottish Borders (UK) over 3 days in July 1999, and on single days in October 1999, April 2000, and August 2000. Cores were separated into four 5-cm depths to examine vertical spatial distribution. The 0–5-, 5–10- and 10–15-cm samples represented organic horizons whilst the 15–20-cm depths were from a mineral horizon. The potential metabolic activities were analysed using BIOLOG-GN plates, whereas genotypic diversity was evaluated using molecular profiling of amplified 16S rRNA and 16S rDNA gene fragments (denaturing gradient gel electrophoresis (DGGE)). BIOLOG-GN analysis revealed decreased substrate utilisation in the lowest depths, which was coupled with changes in the DNA and RNA DGGE profiles. Seasonal variation was pronounced in the 5–10-cm and 10–15-cm organic horizons for the July samplings whilst the 15–20-cm depths appeared more stable. Potential factors influencing the observed changes in bacterial communities resulting from soil depth and sampling time are discussed.