The use of porcine organs is being developed as a means to alleviate the shortage of human organs for transplantation. Recommendations have been published for the microbiological specifications of organ-source pigs to reduce the possibility of a microorganism from pigs being inadvertently transferred to the recipient of the xenograft. The pseudorabies virus (PRV), porcine cytomegalovirus (PCMV), and porcine circovirus (PCV) are infectious agents in pigs that are considered to be of significance for the microbiological safety of xenotransplantation. A multiplex polymerase chain reaction (mPCR) was developed to detect and differentiate among PRV, PCMV, and PCV. The sensitivities of the multiplex PCR were 10(2.5) TCID(50)/ml for PRV, 10(1.8) TCID(50)/ml for PCMV, and 10(1.8) TCID(50)/ml for PCV. The lowest viral concentrations detected by single PCR were 10(1.5) TCID(50)/ml for PRV, 10(1.0) TCID(50)/ml for PCMV, and 10(1.4) TCID(50)/ml for PCV2. Non-specific reactions were not observed when other viruses, bacteria, and Vero cells were used to assess the multiplex PCR. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens collected for xenotransplantation.