The nitrogen cycle controls the availability of nitrogen to plants and other soil organisms. Although great progress has been made in understanding the microorganisms involved in the nitrogen cycle, much is still unknown due to difficulties in cultivating these microbes in the lab. A variety of molecular tools have contributed to the study of these microbes, but there are still limitations to many of these techniques. In an attempt to minimize these limitations we are applying a new strategy to the study of nitrogen cycling organisms. This strategy combines two powerful molecular techniques, microarray analysis and multiplex PCR, to allow parallel amplification and detection of a large number of different genes within a short period of time. We designed a total of 41 primers targeting specific variants of nitrogen cycling functional genes nifH, amoA, nirS, nirK, and nosZ. The specificity of each primer pair was confirmed with DNA from appropriate reference organisms and with DNA from environmental samples. Forty nine oligonucleotide probes, specific for each of the amplicon products, were designed and printed on gel-based DNA microarrays. The microarrays were hybridized with amplicons produced by PCR using Texas Red labeled primers, both in a symmetric and asymmetric manner. Preliminary experiments determined that oligonucleotide concentrations of 0.25mM and 0.1mM produced significantly higher hybridization signals than 0.01mM, that asymmetric PCR yielded significantly higher hybridization signals than symmetric PCR, and that hybridization of asymmetric PCR amplicons at room temperature yielded specific detection of each of the functional gene variants. We have tested a six-plex PCR and have been able to identify amplification from 5 of the 6 primers used asymmetrically. This approach (multiplex PCR prior to microarray hybridization) has the potential to significantly decrease the time necessary for the analysis of functional guild diversity in environmental samples.