The populations of N2-fixing and denitrifying bacteria in an acid forest soil near Cologne were characterized by gene probing. The DNA isolated from the soil for this purpose was suitable for DNA–DNA hybridization using 0.4–0.7-kb probes targeting denitrification enzymes, dinitrogenase reductase (nifH) and eubacterial 16S rRNA. The densitometrical comparison of band intensities obtained in these Southern hybridizations indicated that the highest number of total bacteria, of denitrifying and N2-fixing microorganisms always occurred in the upper (∼5 cm) soil layer. The concentration of all these organisms decreased in parallel with the soil depth. The soil investigated was rich in nitrate in all layers, and the availability of nitrate apparently did not govern the distribution of denitrifying and N2-fixing bacteria in this soil. Soil cores investigated in the laboratory formed N2O on addition of nitrate irrespective of the presence of C2H2. Hybridization intensities, with a gene probe for the 16S rRNA, and MPN numbers were generally higher in soil samples taken from the roots of plants than in the bulk soil. There was no selective enrichment of denitrifying or N2-fixing bacteria at the roots. The data obtained by hybridizing isolated soil DNA generally matched previous results obtained with culturable bacteria.