Nonradioactive slot blot hybridization assays were established for the detection of porcine parvovirus (PPV), using either a digoxigenin-labeled DNA probe or a biotinylated RNA probe. All probes were prepared from a 3.3-kb Pst1-EcoR1 DNA fragment of the NADL8 isolate of PPV. The sensitivity and specificity of the probes in a slot blot system were evaluated in comparison with a 32P-radiolabeled RNA probe. Using an anti-digoxigenin alkaline phosphatase detection system, at least 1 ng of viral replicative form (RF) DNA, or the equivalent of 100 plaque forming units (PFU) of infectious virus, could be detected by the digoxigenin-labeled DNA probe. When the biotinylated RNA probe and a strepavidin-alkaline phosphatase detection system were employed, 0.1 ng of RF DNA, or the equivalent of 10 PFU of infectious virus, were detected, comparable to the sensitivity of the 32P-radiolabeled RNA probe. Hybridization was not observed with control DNA samples extracted from swine testicle cells, porcine kidney (PK-15) cells, uninfected mixed swine fetal tissue, or from an unrelated DNA virus (pseudorabies virus) infected PK-15 cells. Different isolates of PPV, namely NADL8, NADL2, KBSH, and Kresse, reacted on an equimolar basis in sensitivity and specificity to the biotinylated probe. Extraction of DNA directly on the filter membrane (direct filter hybridization) was employed in an attempt to reduce processing time by eliminating DNA extraction steps. Direct filter hybridization was indeed less time consuming; it was also comparable in sensitivity and specificity to those methods employing purified DNA.