The characteristics of the transport systems of L-alanine in lactating mouse mammary gland and their regulation by lactogenic hormones have been studied. L-alanine uptake was mediated by three Na+-dependent and one Na+- independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. Cl− dependency has been established for system A. The other two Na+-dependent systems, which we have named BCl−-dependent and BCl−-independent, are described for the first time. These are systems with broad specificity and were distinguished on the basis of inhibition analysis, Cl− dependency and the effect of preloading mammary tissue with amino acids. The Na+-independent route was identified as system L, which operates independent of Cl−. The A, L and BCl−-independent transport systems were upregulated in pregnant mouse mammary tissue cultured in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin). Insulin alone also upregulated systems A and L to some extent in pregnant mouse mammary tissue. BCl−-dependent activity was not detected in pregnant mouse mammary tissue and was not induced by lactogenic hormones in vitro.