In order to study an antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax₁ antigen, we generated and characterized a panel of rat anti-tax₁, monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I⁺ ) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax₁ antigen in HTLV-I⁺ cell lines and a recombinant tax₁ antigen, PX141 (containing entire tax₁ polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax₁ antigen, XD59 (tax₁ amino acids 180-338); (3) None of them reacted with another truncated tax₁ antigen, XD128 (tax₁ amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax₁-related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax₁ antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax₁ antigen. An antigenic structure of the tax₁ antigen deduced from reactivity of a panel of anti-tax₁ MAbs including the present rat MAbs is discussed.