We analyze the role of 5'HS2 of the mouse beta-globin LCR in the transcriptional and developmental regulation of beta-globin gene expression. Previous studies have shown that the human beta-globin gene behaves as an adult gene in transgenic mice, being expressed in fetal liver and bone marrow-derived erythroblasts but not in yolk sac-derived embryonic erythroid cells. We show that linkage of mLCR5'HS2 to a human beta-globin gene alters this pattern of expression during ontogeny, resulting in expression of the linked beta-globin gene at all stages of murine erythroid development. Expression was independent of integration position and correlated with transgene copy number. Our results provide the first test of a phylogenetically homologous LCR in transgenic mice and demonstrate evolutionary conservation of both developmental and transcriptional potentiation functions between mammalian beta-globin LCRs.