Virus isolation (VI), immunohistochemistry (IHC), fetal serology, and reverse-transcription polymerase chain reaction assay (RT-PCR) were performed on samples from 107 fetuses comprising 10 litters taken from sows experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). In addition to comparing the relative sensitivity and specificity of each test, RT-PCR was evaluated with respect to the relative suitability of thoracic fluids and tissues as samples, the effects of autolysis, and the effects of pooling of fetal specimens. VI, IHC, and fetal serology identified PRRSV infection in 48.6%, 23.4%, and 14.9% of 107 fetuses, respectively, and identified at least 1 infected fetus in 10, 10, and 5 of 10 litters, respectively. In utero death with autolysis reduced the test efficacy of all 3 methods. Fetal thoracic fluid and tissues were equally suitable for RT-PCR detection of PRRSV. Pooling fetal tissues or fluids from VI-positive animals with comparable material from negative controls had no detrimental effect on RT-PCR results when evaluated at dilutions of 1:1, 1:2, 1:4, and 1:8. The results of RT-PCR testing were positive in 100%, 94.4%, and 83.3% of VI-positive specimens allowed to autolyze at 4, 21, or 37 C, respectively, for 24, 48, and 96 hours. Compared with the other testing modalities, RT-PCR appeared to be impacted the least by the adverse effects of autolysis.