We have previously reported on the identification of a monoclonal antibody (mAb) directed against the human CD4 antigen which is capable of activating CD4+ peripheral blood T cells in the absence of other stimuli. In the present study, we extended these findings by demonstrating that the mAb, termed B66, was able to induce the production of interleukin-2 in murine T-cell hybridoma transfectants expressing the human CD4 glycoprotein. Moreover, we found that incubation of Jurkat cells with mAb B66 resulted in the nearly complete disappearance of both CD4 and CD3 from the cell surface, whereas modulation of CD4, but not CD3, was observed after incubation with a non-stimulatory anti-CD4 mAb. Similar results were obtained in modulation experiments using human CD4-expressing murine transfectants. It is thus conceivable that the stimulatory activity of anti-CD4 mAb B66 may be associated with an effect on the CD3 molecular complex. While the biochemical basis for the unique stimulatory activity of mAb B66 has yet to be defined, these findings provide direct evidence that cross-linking of CD4 alone may cause T-cell activation, thus supporting the notion that this glycoprotein can transduce independent positive signals upon binding to class II major histocompatibility complex molecules.