A microcytotoxicity assay for murine H-2 antigens has been developed. The assay is performed in a microdroplet under mineral oil using 3,000 splenic lymphocytes as target cells, microliter quantities of antisera, and either rabbit or guinea pig complement. Cell death is determined by eosin uptake using phase contrast microscopy. The assay can be completed in 2–3 hr and uses minimal amounts of antisera. The method can also be used to detect microgram quantities of soluble H-2 antigens by their ability to inhibit cytotoxicity.