It is anticipated that the rate of sexual transmission of viruses could be substantially decreased by the use of topical chemical barrier methods. Chemical modification of bovine (β-lactoglobulin (β-LG), the major protein of whey, led to the generation of a potent inhibitor (designated 3HP-β-LG) of human immunodeficiency virus type 1 (HIV-1) infection which was also active against herpesviruses. Compounds intended for topical application to prevent sexual transmission of viruses need to maintain their antiviral activity at pH <<7, corresponding to an acidic vaginal environment, and in the presence of seminal fluid. Results presented here show that the binding of 3HP-β-LG to the CD4 receptor for HIV, involved in the anti-HIV-1 activity of this compound, decreases with decreasing pH. The presence of seminal fluid also decreased the binding of 3HP-β-LG to CD4 and diminished the inhibitory effect of the compound on CD4-gp120 binding. 3HP-β-LG was shown to bind Zn++, and the inhibitory effect of seminal fluid could be substantially diminished by chelating Zn++ with ethylenediaminetetraacetate. Saliva had no effect on 3HP-β-LG binding to CD4 or on its interference with gp120-CD4 binding. The decreased 3HP-β-LG-CD4 binding and the concomitant reduction of gp120-CD4 binding inhibition by 3HP-β-LG at low pH and in the presence of seminal fluid could be compensated for by an increase of the 3HP-β-LG concentration and by adding Zn++ chelators to 3HP-β-LG. These results provide a background for the design of 3HP-β-LG formulations for topical use.