The spatial heterogeneity of bacterial populations at a shallow-water hydrothermal vent in the Aegean Sea close to the island of Milos (Greece) was examined at two different times by using acridine orange staining for total cell counts, cultivation-based techniques, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. Concurrent with measurements of geochemical parameters, samples were taken along a transect from the center of the vent to the surrounding area. Most-probable-number (MPN) counts of metabolically defined subpopulations generally constituted a minor fraction of the total cell counts; both counting procedures revealed the highest cell numbers in a transition zone from the strongly hydrothermally influenced sediments to normal sedimentary conditions. Total cell counts ranged from 3.2 x 10(5) cells ml(-1) in the water overlying the sediments to 6.4 x 10(8) cells g (wet weight) of sediment(-1). MPN counts of chemolithoautotrophic sulfur-oxidizing bacteria varied between undetectable and 1.4 x 10(6) cells g(-1). MPN counts for sulfate-reducing bacteria and dissimilatory iron-reducing bacteria ranged from 8 to 1.4 x 10(5) cells g(-1) and from undetectable to 1.4 x 10(6) cells g(-1), respectively. DGGE revealed a trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone to a community dominated by few populations close to the center of the vent. Temperature was found to be an important parameter in determining this trend. However, at one sampling time this trend was not discernible, possibly due to storm-induced disturbance of the upper sediment layers.