In situ biological denitrification has been proposed as an important metabolic activity in the remediation of nitrate-contaminated groundwater. In this study, the effects of fumarate, an electron donor for biological denitrification, on the in situ denitrifying activity were determined by using three types of single-well push-pull tests; transport, biostimulation and activity tests. During the tests, changes in microbial community composition were also investigated using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. Transport test demonstrated that non-reactive tracer and biologically reactive solutes behaved similarly. A biostimulation test was conducted to stimulate the denitrifying activities of native microorganisms, which were monitored by detecting the simultaneous production of CO(2) and drastic degradations of both nitrate and fumarate after the injection of fumarate as an electron donor and/or carbon source, with nitrate as an electron acceptor. A phylogenetic analysis suggested that the taxonomic affiliation of the dominant species before biostimulation was γ-Proteobacteria, including Acinetobacter species and Pseudomonas fluorescens, while the dominant species after biostimulation were affiliated with β-Proteobacteria, cytophaga-Flavobacterium-Bacteroides and high G+C gram-positive bacteria. These results suggest that the analyses of groundwater samples using a combination of single well push pull tests with DGGE can be applied to investigate the activity, diversity and composition shift of denitrifying bacteria in a nitrate-contaminated aquifer.