The V3 loop of the gp120 envelope glycoprotein contains the principal neutralizing determinant of HIV-1. Many serological studies have been carried out to assess the reactivity of HIV-1 infected individuals against V3 loop synthetic peptides from different HIV-1 subtypes. V3 directed serology has also been used to demonstrate the association between ELISA reactivity and progression to AIDS in HIV patients, and to study the reactivity against the V3 region in sera from vaccinated animals and human volunteers. The advantage of the use of bovine serum albumin (BSA) conjugated V3 peptides over free V3 peptides for ELISA is described. 15 meric V3 peptides representing several HIV-1 isolates were synthesized, chemically coupled to BSA, and used to coat ELISA microplates. Conjugated peptides were compared with free peptides for the detection of anti V3 antibodies in the sera from rabbits immunized with V3 containing chimeric proteins and from HIV-1 infected individuals. No differences in reactivity against free or BSA-peptide were found for most rabbit sera, however human plasma recognized preferentially the BSA conjugated peptides. Although technically more complex, ELISA with BSA coupled V3 peptides is more sensitive and appropriate for serological studies of HIV-1 infected persons.