T lymphocyte clones were derived by micromanipulation from an MLC between a stimulator and responder matched for class I but mismatched for class II HLA antigens; among the possible stimulating antigens were DR4 and Dw4. Two of the clones, when tested for cytotoxicity on a panel of DR4 positive and negative targets, appeared to recognize a determinant closely associated with Dw4, but did not lyse, with one exception, targets expressing other DR4 associated Dw specificities or DR4 negative targets. Blocking studies, using monoclonal antibodies directed against monomorphic epitopes on class I or class II molecules, revealed that the cytotoxic activity of these clones was strongly inhibited by an anti-class II (L-243) but not by an anti-class I (w6/32) monoclonal antibody. Both clones were T3+, T4+, T8-. These findings show that cytotoxic T cell clones, (directed against class II antigens), may have specificity that correlates with a T lymphocyte-defined LD/Dw determinant. The blocking experiments using monoclonal antibodies give further support to the idea that these clones recognize determinant(s) on a class II molecule. The cell surface phenotyping results are in agreement with previous reports that most anti-class II cytotoxic clones have a T4+ T8- phenotype.