Murine anti-human class II antibodies were shown to cross-react with polymorphic determinants of murine class II antigens. The cross-reacting antibodies were raised in B10.S(9R) mice by immunizing with human nylon wool adherent cells (Ad cells) from peripheral blood leukocytes. The B10.S(9R) anti-human Ad cell antiserum bound to the molecules consisting of two chains with molecular weights of 35K and 28K dimers which were purified with a lentil-lectin column. The B10.S (9R) anti-human class II antiserum was also revealed to contain two distinct cross-reacting antibodies with polymorphic determinants of murine class II antigens coded for by the I-A subregion of the H-2. One is specific for a determinant of class II molecules coded for by I-Ab,d,q, and the other seems to be specific for class II molecules coded for by I-Aa,k,r.