Pseudorabies virus (PRV) propagated in rabbit kidney-derived RK-13 cells (PRV-RK) was neutralized by serum obtained from specific pathogen-free pigs through the activation of complement. The virus-neutralizing activity of swine serum was lost after treatment with ethylene glycol-bis-aminoethylether-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). Anti-C1q and anti-IgM antibodies also inhibited virus-neutralizing activity. Though IgG-depleted swine serum neutralized PRV, IgM and IgG-free swine serum lost virus-neutralizing activity. Pre-incubation of swine serum with RK-13 cells, but not with swine kidney-derived CPK cells, at 4°C eliminated the virus-neutralizing activity to PRV-RK. Results indicated that swine serum contained natural IgM against an antigen(s) on the RK-13 cell surface and that this surface antigen was integrated into the PRV envelope during the budding process. Thus the natural IgM in swine serum reacted with the RK-13 antigen on the viral envelope, activated the complement cascade and neutralized the PRV-RK.