To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a Salmonella-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonella organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100% compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.