In an effort to derive monoclonal antibodies (mAb) which can induce production of macrophage-activating factor (MAF) by cloned murine cytolytic T lymphocyte (CTL) lines, we have fused spleen cells from a rat immunized with a CTL clone with the nonsecreting mouse myeloma X63-Ag8.653. Three mAb (designated I-22, III-5 and V-8) were found to stimulate MAF production by the immunizing CTL clone and (with a single exception) two other unrelated CTL clones. However, none of these mAb inhibited the cytolytic activity of the clones. Immunoprecipitation studies indicated that the three mAb reacted primarily with a 25-30-kDa protein which could not be distinguished from that precipitated by either a reference anti-Thy-1.2 mAb or a polyclonal rabbit anti-Thy-1 antiserum. Moreover, competition binding experiments demonstrated that the three mAb competed with each other and with the reference anti-Thy-1.2 mAb. Flow cytofluorometric analysis of the strain distribution of the molecules defined by the mAb revealed that two of the antibodies (I-22 and III-5) were directed against nonpolymorphic determinants of Thy-1, whereas V-8 mAb reacted only with Thy-1.2+ lymphocytes. One of the mAb (III-5) was also able to stimulate proliferation and interleukin 2 secretion by normal splenic T cells. Since mAb directed against a number of other surface structures on CTL clones did not stimulate MAF production, it thus appears that Thy-1 (or molecules associated with Thy-1) may play a functional role in T lymphocyte triggering.