A pseudorabies virus (PRV) nucleocapsid based screening ELISA (NC-ELISA) was developed. The NC-ELISA was compared to a commercial PRV screening ELISA. Antibody to PRV was detected by the NC-ELISA between day 14 and 21 in 5 pigs after intranasal exposure to 10[superscript]4 PFU of virus and was still detected on day 42. The NC-ELISA also detected antibody in 23 of 24 naturally infected pigs. The commercial ELISA detected antibody 1 week earlier than NC-ELISA in experimentally infected pigs but didn't detect antibody in 3 naturally infected pigs identified by NC-ELISA. Radioimmunoprecipitation confirmed the presence of PRV specific antibody in these pigs. Nucleocapsid specific antibody responses of 10 PRV glycoprotein subunit-vaccinated were monitored for 113 days after exposure to a low dose (10[superscript]2.3 PFU) of PRV. Nucleocapsid specific antibody responses remained below the positive threshold before challenge but increased dramatically following virus exposure. Mean NC-ELISA OD's were above the positive threshold on day 113 p.c;The potential of two in vitro translation products representing truncated pseudorabies virus (PRV) major nucleocapsid proteins was also studied. Two cDNA clones designated p462A and p462D, containing 800 and 910 bp cDNAs respectively were identified by hybridization with [superscript]32P-labelled PRV genomic BamHI-D fragment. Northern blot analysis showed that [superscript]32P-labelled insert cDNA probes 462A and 462D hybridized to 1.5 and 4.4 kb RNAs respectively in total RNA from PRV infected MDBK cells. In vitro transcription and translation of cDNA 462A yielded a polypeptide doublet with a molecular weight of 19 and 21k. Similarly, cDNA 462D produced a single polypeptide with a molecular weight of 24 k. Pseudorabies virus infection was detected by radioimmunoprecipitation in 21 of 23 pigs when reactions with both in vitro translation products were considered. Sera from 15 of 23 PRV infected pigs immunoprecipitated the 462A polypeptide doublet, while sera from 16 of 23 pigs immunoprecipitated the 462D polypeptide. In addition, sera from 4 of 4 PRV low dose challenged (10[superscript]2 PFU) pigs immunoprecipitated both translation products beginning at day 14 p.i. and continuing to the last day tested, day 60 p.i.