Dietary supplementation of L‐arginine as a mechanism to enhance cellular immune response (T lymphocytes), has slowly gained approval, and appears especially important during critical illness. Despite its clinical use, little is known as to the direct effects of L‐arginine on the different T lymphocyte subpopulations. Lymphocytes were harvested from spleens of C57 Bl/6 mice, and proliferation was induced with anti‐CD3 in the presence of different concentrations of L‐arginine ranging from 0 to 1000 μmol/L. Flow cytometry was used to evaluate the effect of L‐arginine on T lymphocyte subpopulations. Interleukin‐2 production was measured by ELISA and gene expression by RT‐PCR. L‐Arginine at or greater than 100 μmol/L significantly enhanced anti‐CD3 stimulated T lymphocyte proliferation (p =.01). L‐Arginine was essential for adequate T lymphocyte (CD3+) cellular maturation (p =.01). Proliferation of Helper T cells (CD4+) was not dependent on L‐arginine. In contrast, Cytotoxic T cells (CD8+) showed a dose dependent proliferation in response to L‐arginine (p =.01). Of the CD8+ cells, an increase in the CD45RA negative CD8 positive (memory) T cell subpopulation was observed with the addition of L‐arginine. In addition, the number of cell surface CD8 receptors (CD8R) and CD3 receptors (CD3R) increased in the presence of L‐arginine (p =.01, p =.04). Interleukin‐2 receptor (IL‐2R) expression was not up‐regulated by L‐arginine. L‐Arginine modestly increased IL‐2 production and had pronounced effects on its disappearance from the culture media (p <.0001). Interleukin‐2 mRNA expression was not dependent on L‐arginine. The requirements for L‐arginine for the proliferation of CD3 stimulated T lymphocytes vary widely, and have to be taken into account when studying the mechanism of how L‐arginine enhances cellular proliferation. L‐Arginine may increase cellular proliferation by increasing specific receptor expression and the utilization of interleukin‐2. (Journal of Parenteral and Enteral Nutrition 25:23–29, 2001)