Four rat mAb directed against mouse IL-5 have been characterized by their ability to remove and neutralize mouse IL-5 activity in various bioassays. All four mAb absorbed IL-5 activity from solution. Although all were able to neutralize mouse IL-5 bioactivity, two were significantly more effective. These two were also able to neutralize the activity of mouse IL-5 delivered to B cells during a cognate-linked interaction with a Th cell clone. A two-site sandwich ELISA specific for mouse IL-5 was developed by using pairs of mAb. The mouse IL-5 ELISA is capable of detecting natural or mouse rIL-5 in supernatants, crude bacterial lysates, and high concentrations of mouse serum, and has a detection limit of less than 20 pg. Two of these antibodies cross-reacted with and neutralized human rIL-5, and one of these was used for development of an ELISA for human IL-5.