The applicability of the DNA sandwich hybridization method to detection of bacterial DNA from crude samples is demonstrated using Escherichia coli as a model. In sandwich hybridization the sample DNA mediates the binding of a labeled probe DNA fragment to a second DNA bound on filter. For this study the DNA reagents were prepared by subclonings from a recombinant plasmid containing the E. coli K-12 ompA gene and an adjacent fragment of E. coli DNA. The 5' half of the ompA gene (738 base pairs) in pBR322 served as the filter reagent. The 3' half of the ompA gene (300 base pairs) and the adjacent 1,500 base pairs of E. coli DNA were cloned into the single-stranded phage vector M13mp7, and the resulting recombinant phage DNA was labeled with 125I and used as probe in the hybridizations. For maximal hybridization the DNA reagents had to be present in excess of the sample nucleic acid, which was preferably fragmented before testing. In the optimized test, 3 X 10(6) molecules of E. coli DNA from lysed cells were detected by an overnight reaction; the sensitivity of the test was not affected by the presence of 10(9) unrelated bacteria. With the ompA reagents, all members of the family Enterobacteriaceae tested were detected even if the sensitivity was decreased as compared with that for the homologous bacteria. With all other bacteria, including aerobic and anaerobic species of clinical importance, the test was negative. These findings suggest that it may be possible to find group-specific reagents to be used in diagnostic bacteriology.