A germ line null mutation of the prolactin receptor gene has been produced by replacing exon 5 with the Tk-NEO³ cassette. Heterozygous females showed almost complete failure of lactation following their first pregnancy when mated at 6–8 weeks of age. The severity of this phenotype was reduced when heterozygotes were mated at 20 weeks and was absent following a second pregnancy when assessed by pup survival, although a longer lag time prior to weight increase at normal rates was seen in these litters. Histological and whole mount analysis of virgin mammary glands showed that heterozygous glands were smaller in size due to a less developed ductal structure with fewer branch points. Virgin knockout glands were small, composed of sparse, large, poorly branched ducts. This work provides an ideal model to further study the role of the prolactin receptor and its ligands in mammary development and physiology.