We have isolated and characterized 8 mAb against human rIL-2. All recognize nonglycosylated rIL-2 in liquid phase with similar affinities (Kd approximately 1 nM). Based on the epitopes of the IL-2 molecule that they recognize and their pattern of reactivity against glycosylated and non-glycosylated IL-2, they have been classified into four groups. The first group of anti-IL-2 mAb (2C4, 19B11 and 12C2) inhibits IL-2 binding to p70 IL-2R, while the second one (16F11, 18E1 and 2A4) prevents its binding to p55 IL-2R. These two groups neutralize IL-2 activity in a T cell proliferation assay equally well, due to their similar inhibition of IL-2 binding to high affinity IL-2R. Two mAb, 3H9 and 17F4, recognize separate epitopes on IL-2 molecule, are poor inhibitors of IL-2 binding, and they are inefficient in the neutralization of its biological activity; they have been assigned to the third and fourth groups, respectively. These results show that mAb from the first and second group recognize two epitopes of the human IL-2 molecule which probably overlap the p70 IL-2R and p55 IL-2R binding sites, respectively. In addition, these areas together form the high affinity IL-2R binding site. The two mAb from the third and fourth group recognized epitopes of IL-2 not directly involved in IL-2 binding to its receptor. All eight mAb anti-human IL-2 recognize murine IL-2 and with the exception of one, 17F4 mAb are also able to neutralize it in a T cell proliferation assay. The relationship between the structure and the function of the IL-2 molecule is discussed.