Although the etiology of porcine proliferative enteritis is not understood, the consistent presence of intracellular Campylobacter-like organisms (CLOs) in proliferating pig intestinal epithelial cells suggests that the organism is involved in the disease process. In order to obtain information about this organism, we generated and characterized specific DNA probes to the intracellular CLO which was purified without culturing. Intracellular CLOs were isolated from mucosa by homogenization, filtration, and absorption to wheat germ agglutinin-Sepharose. The DNA was purified, and a CLO genomic library was constructed. The specificity of recombinant plasmids was confirmed by both dot blot hybridization and Southern analysis of normal and diseased mucosa, as well as of a variety of Campylobacter species. Several of the CLO-specific probes hybridized with porcine mucosa obtained from pigs with proliferative enteritis but not with nondiseased mucosa. The probes hybridized equally with mucosa or DNA obtained from each of the two clinical forms of proliferative enteritis, i.e., proliferative hemorrhagic enteropathy and porcine intestinal adenomatosis. The CLO-specific probes failed to hybridize with any of the commonly isolated porcine Campylobacter species, including Campylobacter hyointestinalis, C. mucosalis, and C. coli. Therefore, the intracellular CLO of porcine proliferative enteritis may be an as yet unidentified or uncultured species.