A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.