₂-antiplasmin (₂-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine ₂-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with theneomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated ₂-AP allele was observed, and homozygous deficient (₂-AP^−/−) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of ₂-AP mRNA in kidney and liver from ₂-AP^−/− mice, in contrast to wild-type (₂-AP^+/+) mice. Immunologic and functional ₂-AP levels were undetectable in plasma of ₂-AP^−/− mice, and were about half of wild-type in heterozygous littermates (₂-AP^+/−). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for ₂-AP^+/+, ₂-AP^+/−, and ₂-AP^−/− mice. After amputation of tail or toe tips, bleeding stopped spontaneously in ₂-AP^+/+, as well as in ₂-AP^+/− and ₂-AP^−/− mice. Spontaneous lysis after 4 hours of intravenously injected ¹²⁵I-fibrin–labeled plasma clots was significantly higher in ₂-AP^−/− than in ₂-AP^+/+ mice when injecting clots prepared from ₂-AP^+/+ plasma (78% ± 5% v 46% ± 9%; mean ± SEM, n = 6 to 7; P = .02) or from ₂-AP^−/−plasma (81% ± 5% v 46% ± 5%; mean ± SEM, n = 5; P = .008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in ₂-AP^−/− mice, as compared with ₂-AP^+/+ mice (P ≤ .005). Thus, ₂-AP^−/− mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.