A bovine herpesvirus 1 (BHV-1) gIII deletion mutant (gIII-) was produced by means of recombinant DNA that retained the ability to replicate in cell culture. However, the gIllmutant was functionally defective, showing impaired attachment to permissive cells, a delay in virus replication, and reduced extracellular virus production. The attachment defect exhibited by the gIlmutant is an indication of the role played by glll in the normal infection process. This was shown by dramatically decreased binding of radiolabelled glllvirus to permissive cells and a slower adsorption rate, as measured by plaque formation, than the wild-type (wt) virus. Furthermore, treatment of the glllvirus with neomycin increased virus adsorption and plaque formation by severalfold, whereas neomycin treatment had no effect on the wt virus. This observation showed that the glllmutant was strictly defective in adsorption but fully competent to produce productive infections once induced to attach. The glllmutant showed greater sensitivities than did the wt virus to anti-gI and anti-gIV antibody-mediated neutralization. Analyses with panels of monoclonal antibodies to gI and gIV revealed that the epitopes gI-IV and gIV-III were the main targets for enhanced neutralization. This provided evidence that gI and gIV may also participate in virus attachment. Finally, when affinity-purified gI, glll, and gIV were tested for their ability to inhibit virus adsorption, gIlI had the most pronounced inhibitory effect, followed by gI and then gIV. glll was able to completely inhibit wt virus adsorption, and at a high concentration, it also partially inhibited the glllmutant. gI and gIV inhibited wt and glllmutant adsorption to a comparable extent. Our results collectively indicate that glll plays a predominant role in virus attachment, but gI and gIV also contribute to this process. In addition, a potential cooperative mechanism for virus attachment with these three proteins is presented.