Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.