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Microorganisms (e.g., prokaryotes, fungi) are food sources for soil nematodes, but they can also be potential mutualists or pathogens. Understanding the linkages between microorganism and invertebrate diversity in soils requires the ability to distinguish between these microbial roles. We tested the potential of a taxon-specific fluorescent in situ hybridization (FISH) procedure for identifying and localizing microbial rRNA within the bodies of soil nematodes. Our objective was to determine whether the rate of digestion permitted detection and identification of food-source nucleic acids within the nematode digestive system (i.e., pharynges, intestines) before their breakdown. First, using laboratory cultures of Caenorhabditis elegans maintained on Escherichia coli, we were able to localize bacterial rRNA throughout the nematode pharynx with the universal bacterial-probe EUB338, although never in the intestines. Second, we applied the fungal rRNA probe FR1 to Aphelenchus avenue cultured on the fungus Rhizoctonia solani. We were unable to detect fungal rRNA within these nematodes, and it appears that this material may be digested rapidly. Next, we applied our technique to nematodes extracted directly from soils. We were able to localize bacterial rRNA within the pharynges of bacterial-feeding species of nematodes from desert soils. We also localized archaeal rRNA using the probe ARC344. Finally, application of EUB338 to desert soil nematodes revealed the presence of bacteria in the intestines of some nematodes and within the ovary of a single nematode. This technique has great potential for use in understanding the feeding behavior of bacterial-feeding soil nematodes and in studies of nematode:bacterial relationships. (C) 2010 Elsevier Ltd. All rights reserved.