Interleukin-2 (IL-2) activates extracellular signal-regulated protein kinase (ERK) within immune cells. To examine the profile of phosphorylated ERK (p-ERK) in IL-2 stimulated immune cells of normal donors and patients receiving IL-2 therapy, we developed a dual parameter flow-cytometric assay. An analysis of PBMCs stimulated with IL-2 indicated that IL-2 exposure induced p-ERK in CD56bright NK cells and CD14+ monocytes, but not in CD3+ T cells or CD21+ B cells. CD3+ T cells that were induced to express functional high-affinity IL-2R did not exhibit enhanced p-ERK following IL-2 treatment. Measurement of p-ERK within PBMCs from cancer patients 1 h following their first dose of IL-2 revealed a complete absence of circulating NK cells, consistent with earlier observations. However, the total number of circulating CD14+ monocytes increased in these samples and 97% of these cells exhibited ERK activation. p-ERK was not observed in T cells post-IL-2 therapy. Analysis of PBMCs obtained 3 weeks post-IL-2 therapy revealed high-p-ERK levels in CD56bright NK cells in a subset of patients, while levels of p-ERK returned to baseline in monocytes. These studies reveal an effective method to detect ERK activation in immune cells and demonstrate that IL-2 activates ERK in a subset of NK cells and monocytes but not T cells.