The CD6 glycoprotein is expressed primarily on lymphocytes and conveys co-activating signals to T cells, but its exact function and ligand(s) are unknown. A novel mAb, termed UMCD6, was demonstrated to recognize CD6 by immunoprecipitation, Western blotting, and reactivity with COS cells transfected with CD6 cDNA. UMCD6 was mitogenic for T cells and was strongly synergistic with phorbol ester in inducing T cell activation. UMCD6 enhanced the autologous mixed lymphocyte reaction as previously observed with another anti-CD6 mAb, anti-T12. The activating effects of UMCD6 were more striking than those of other anti-CD6 mAbs and encompassed all of the diverse stimulatory properties previously reported for other anti-CD6 reagents. However, neither UMCD6 nor other anti-CD6 antibodies alone or in combination with phorbol ester or IL-2 were able to induce thymocytes to proliferate. Stimulation by UMCD6 is dependent on accessory cell function in a manner not accounted for simply by antibody crosslinking. UMCD6 did not induce an increase in cytoplasmic free Ca²⁺, but the CD6 activation pathway appears to involve protein kinase C. UMCD6 and a panel of seven other anti-CD6 mAbs were used in a series of experiments to define four discrete epitopes of CD6 using the criteria of antibody cross-blocking, reactivity on reduced Western blots, and resistance to controlled V8 protease digestion. The functional mAbs UMCD6, 2H1, and antl-T12 each recognized a different epitope. Taken together, the results of these studies strongly reinforce the hypothesis that CD6 plays a significant and distinct role in T cell activation, and suggest that multiple regions of CD6 may be functionally active.