The human beta-globin locus control region (LCR) contains chromatin opening and transcriptional enhancement activities that are important to include in beta-globin gene therapy vectors. We previously used single-copy transgenic mice to map chromatin opening activity to the 5'HS3 LCR element. Here, we test novel hybrid globin genes to identify beta-globin gene sequences that functionally interact with 5'HS3. First, we show that an 850-base pair (bp) 5'HS3 element activates high-level beta-globin gene expression in fetal livers of 17 of 17 transgenic mice, including 3 single-copy animals, but fails to reproducibly activate Agamma-globin transgenes. To identify the beta-globin gene sequences required for LCR activity by 5'HS3, we linked the 815-bp beta-globin promoter to Agamma-globin coding sequences (BGT34), together with either the beta-globin intron 2 (BGT35), the beta-globin 3' enhancer (BGT54), or both intron 2 and the 3' enhancer (BGT50). Of these transgenes, only BGT50 reproducibly expresses Agamma-globin RNA (including 7 of 7 single-copy animals, averaging 71% per copy). Modifications to BGT50 show that LCR activity is detected after replacing the beta-globin promoter with the 700-bp Agamma-globin promoter, but is abrogated when an AT-rich region is deleted from beta-globin intron 2. We conclude that LCR activity by 5'HS3 on globin promoters requires the simultaneous presence of beta-globin intron 2 sequences and the 260-bp 3' beta-globin enhancer. The BGT50 construct extends the utility of the 5'HS3 element to include erythroid expression of nonadult beta-globin coding sequences in transgenic animals and its ability to express antisickling gamma-globin coding sequences at single copy are ideal characteristics for a gene therapy cassette.