Nitrogen fixation is often enhanced in the rhizosphere as compared with bulk soil, due to asymbiotic microorganisms utilizing root exudates as an energy source. We have studied the activity and composition of asymbiotic soil diazotrophs following pulse additions of artificial root exudates and single carbon sources, simulating the situation of bulk soil coming into contact with exudates from growing roots. Artificial root exudates and single sugars rapidly induced nitrogen fixation. The population of potential diazotrophs was studied using universal and group-specific nifH polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis. Reverse transcription PCR of nifH mRNA confirmed that phylotypes with an apparently increasing population size also expressed the nitrogenase system. According to our results, the actively nitrogen-fixing population represents only a fraction of the total diazotroph diversity, and the results of group-specific nifH PCR and phylogenetic analysis of cloned nifH and 16S rRNA gene fragments identified active species that belonged to the genus Azotobacter. Rapid changes of transcriptional activity over time were observed, indicating different growth and activation strategies in different Azotobacter strains. Only sugar-containing substrates were able to induce nitrogen fixation, but substrate concentration and the presence of organic acids may have additional selective effects on the active diazotroph population.