Prompted by diagnosis of paratuberculosis in 2 blesbok in 1991 and a Chinese Reeve's muntjac in January, 1992, a survey for paratuberculosis of all ungulates at the San Diego Wild Animal Park was initiated. Fecal samples were collected, weighed and 3gm was placed into 30ml of HPC decontaminant/transport medium at the Park, then sent to the laboratory within 3 days collection. Samples were processed to detect growth of mycobacteria in a radiometric culture system (BACTEC) modified to grow M. paratuberculosis (M. ptb). Most M. ptb isolates were detected after 3-5 weeks of incubation and cultures were declared negative if no growth was detected after 7 weeks. The majority of mycobacterial isolates were identified using a commercial PCR amplified DNA probe for M. ptb (IDEXX Laboratories, Inc.). When the isolates tested negative with this probe, they were tested using a series of RNA probes for other mycobacterial pathogens, M. avium, M. intracellulare, and the TB complex (M. tuberculosis and M. bovis), (AccuProbe, GenProbe, San Diego, CA). Over 80% of the 1500 ungulates at the park were sampled. Over 3,000 fecal and tissue specimens were tested by radiometric culture from January 1, 1992 to December 31, 1993. Seventy-seven isolates of mycobacteria were obtained; 55 were M. ptb. No mycobacterial isolates were found to be in the TB complex. Ten saprophytic mycobacterial species that tested negative with the genetic probes were identified by a reference laboratory. M. ptb was isolated from 28 different species of animals. The majority of these animals lived in one of three adjacent enclosures and M. ptb was isolated from pond water in one of them. As of January 1, 1994, the infection appears limited to roughly one-third of the Park. Infection prevalence in that portion of the park was estimated at 10%. Measures have been instituted to prevent spread of the infection to other parts of the park. In addition, the more heavily infected species of animals, red deer, springbok, muntjac, and blesbok have been removed from the enclosures. Surveillance of animals at the park by fecal culture continues. Movement within and out of the collection is restricted to animals found negative on repeated fecal cultures or from areas/exhibits declared negative.