Porcine reproductive and respiratory syndrome (PRRS) virus infection results in clinically normal, but persistently infected animals. An understanding of the carrier state is necessary for prevention, control and/or elimination of PRRS virus. The objective of this experiment was to estimate the proportion of PRRS virus carriers over time and determine which combination of sample and diagnostic assay could most effectively identify persistently infected animals. Sixty 3-week-old pigs were inoculated with PRRS virus ATCC VR-2332 and followed for up to 105 days post-inoculation (PI). Sixty age-matched animals served as uninoculated controls. Samples (serum, peripheral blood leukocytes, oropharyngeal scrapings, tonsil, bronchoalveolar lavage, lung tissue and tracheobronchial lymph nodes) were collected periodically and tested for evidence of PRRS virus infection by virus isolation (VI), swine bioassay and reverse transcriptase-nested polymerase chain reaction (RT-nPCR). The PRRS virus-specific antibody response was monitored with a commercial enzyme-linked immunosorbent assay (ELISA). Overall, PRRS virus was found in 51 of the 59 (84%) necropsied animals by VI or swine bioassay between 63 and 105 days PI, including 10 of the 11 (91%) of animals at day 105 PI. RT-nPCR on oropharyngeal scrapings was the most effective combination of assay and sample for detecting carriers. There was no significant difference in the antibody response of carrier vs. non-carrier animals. Infectious PRRS virus is present in most pigs the first 105 days following infection. Antibody response, as measured by a commercial ELISA, cannot be used to determine carrier status. RT-nPCR is a useful tool for detection of carriers, but diagnostic sample selection is critical if false negative results are to be avoided.