A mouse monoclonal antibody to rat 5′-nucleotidase (5N 4−2 McAb) was used in the direct anti-determinant rosetting reaction (DARR) to demonstrate the ecto-5′-nucleotidase molecule in preparations of rat lymphocytes. Results indicated that 35.5 ± 7.5% of peripheral blood lymphocytes (PBL), 37.3 ± 4.8% of lymph node cells (LN) and 37.0 + 8.5% of spleen lymphocytes expressed the 5N 4−2 antigen. Depletion studies and mixed rosetting reactions (MRR) showed that the 5N 4−2 antigen was mainly expressed on rat T lymphocytes rather than on B lymphocytes: In fact 59.6 ± 3.2% (in PBL), 76.5 + 0.6% (in LN) and 67.1 ± 1.3% (in spleen) of T lymphocytes exhibited the 5N 4–2 antigen compared to only 26.5 ± 2.6% (in PBL), 34.0 + 2.1% (in LN) and 46.1 + 12.0% (in spleen) of B lymphocytes. As expected a strong association was found between the expression of 5N 4−2 antigen and 5′-nucleotidase enzyme activity on lymphocytes. Both 5N 4–2 positive cells and enzyme activity were preferentially exhibited in the T lymphocyte subpopulation, and 92% of the enzyme activity was observed in a 5N 4−2 antigen positive subpopulation.