A monoclonal mouse antibody, 4-B-5, that reacted with both single-stranded DNA (ssDNA) and double-stranded DNA showed direct reactivity and cell association with mouse thymocytes, as well as peripheral blood mononuclear cells. Absorption of 4-B-5 with peripheral blood mononuclear cells or with mouse thymocytes markedly reduced reactivity with both ssDNA and double-stranded DNA. However, treatment of mouse thymocytes or human T cells with DNase completely eliminated monoclonal anti-DNA reactivity with cells. The cellular reactivity was completely restored when monoclonal anti-DNA was incubated with DNase-treated cells in the presence of normal human serum. Normal human serum was found to contain 574 +/- 639 ng/ml of ssDNA. Coincubation or cell preincubation of mouse thymocytes with ssDNA produced a marked increase in 4-B-5 cell association. These findings indicate that mouse monoclonal anti-DNA can react with and can penetrate both mouse and human mononuclear cells, and that this reactivity may depend on the presence of cell membrane DNA on both types of target cells.