We have used gene targeting to generate relaxin (rlx)-deficient mice. The majority (15 of 17) of homozygous (rlx−/−) mice are fertile and produce normal litters. However their mammary development is deficient; pups are unable to suckle and die within 24 h of birth unless cross-fostered to a wild-type (rlx+/+) foster mother. The nipples of rlx−/− animals do not enlarge significantly during pregnancy, and their histology retains the appearance of the virgin state. Breast parenchyma is somewhat underdeveloped at term even though milk is produced. Mammary ducts become grossly dilated in these animals. Heterozygous (rlx+/−) mice lactate normally. The interpubic ligament does not relax during pregnancy in rlx−/− mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx−/− mice than in wild-type controls.