Members of the EGF family of growth factors play critical roles during normal and neoplastic breast development. EGF family member HRGα is the only HRG1 isoform expressed in the mouse mammary gland and our previous experiments suggest that HRG1 has a unique role in mammary development. To determine the function of HRGα activity during mouse mammary gland development, we generated a HRGα-deficient mouse strain. Unlike mice with HRG1 or isoform specific HRGβ gene deletions, HRGα-null mice survive to adulthood. HRGα-deficient mice display pronounced defects in mammary gland lobuloalveolar development at 17 days of pregnancy and 3 days post-partum. Terminal and lateral ductal alveoli were condensed and alveolar outgrowth during pregnancy was severely impaired. A dramatic reduction in β-casein expression accompanied defective alveolar development in the HRGα-null mice, as determined by in situ hybridization and Northern blot analysis of HRGα-deficient mammary glands at 3 days post-partum. Expression of the milk-protein genes WAP and α-lactalbumin was not adversely affected. In situ incorporation of BrdU demonstrated that epithelial proliferation was significantly curtailed in mammary glands of HRGα-deficient mice at 17 days post-coitus and 3 days post-partum. These results demonstrate that HRGα is an important mammary gland mitogen regulating alveolar development and lactogenesis.