Several polyanionic reagents such as dextran sulfates, heparin sulfates, and negatively charged proteins have been reported to exhibit anti-HIV activity in vitro. Particularly potent inhibition has been reported for the milk protein beta-lactoglobulin (betaLG) on modification by 3-hydroxyphthalic anhydride (3HP). The introduction of multiple negatively charged carboxyl groups along the polypeptide backbone obviously leads to repulsion within the protein molecule and this is likely to affect the specific tertiary, and perhaps also secondary, structure of the protein. We used several biophysical techniques to probe the structural changes that occur on 3HP modification of betaLG. The results suggest that the protein becomes largely unstructured on chemical modification. Although a profound anti-HIV activity was measured for 3HP-betaLG, similar antiviral effects were observed with two other 3HP-modified milk proteins, alpha-lactalbumin and alpha(S2)-casein, but not with the unmodified proteins. Most potent inhibition of HIV-1 replication was obtained with 3HP-modified alpha-lactalbumin, which also demonstrated the least cytotoxicity. These combined results indicate that HIV inhibition is a general property of negatively charged polypeptides and do not support a model in which the negatively charged 3HP-betaLG protein interacts in a structure-specific manner with the CD4 cell surface receptor for HIV-1 entry.