Antisera from sheep immunized with two different recombinant soluble CD4 preparations both exhibited cross-reactive antibodies specific for recombinant gp120s and gp160s prepared in different expression systems in immunoblots and radiobinding assays. The cross-reactivity was broadly mapped to the amino-terminal 70K cleavage fragment of gp120 and more closely identified to residues 204–274 using a series of truncated recombinant gp120s. However, this reactivity could not be detected against virally encoded gp160/120.