In order to reduce the time and cost for screening of pseudorabies virus (PRV)-specific cDNAs, a rapid and inexpensive method was developed that involved subtractive hybridization of the plasmid, which contained cDNA fragment, to PRV genomic DNA which was bound to nylon membranes. Ninety percent of DNA background was subtracted successfully by this method and the eluted DNA in the form of plasmid could be used to transform bacteria directly. Applying this technique, 200 colonies were screened from a cDNA library containing 30000 colonies. Furthermore, 17 colonies containing PRV-specific cDNAs, including PRV43, UL42, gII, DNase, EP0, 11K, gX, and RSP40, were identified from the 200 colonies by colony hybridization, Southern hybridization, and DNA sequencing. Thus, the subtractive hybridization can be used to construct and successfully establish the PRV cDNA library from PRV-infected cells.