Mouse thymidine kinase-negative (TK-) L cells were transformed with concatenates of cloned herpes simplex virus 1 TK DNA and different rabbit beta-globin DNAs in which the globin genes were preceded by native flanking sequences of 14, 66, 76, 425, and 1500 nucleotides.l In all cases, selection for TK+ cell lines led to a high yield of lines producing 5-1500 mature rabbit beta-globin-specific RNA strands per cell. The 5' termini of the transcripts mapped to (i) the "cap" nucleotide, (ii) positions 42 to 48 nucleotides downstream from the cap site, or (iii) positions in the vector DNA preceding the gene. In the case of the gene with only 14 base pairs of 5' flanking sequence, a high level of rabbit beta-globin RNA was produced, but none of the transcripts had the correct 5' end; most of them originated in the vector moiety. With 66 base pairs of 5' flanking sequence, 5% of the 5' termini were correct, and with 76 or more base pairs, 30-85% were correct. The region between 14 and 66 base pairs preceding the cap site contains the Hogness box and appears to be essential for correct initiation of transcription. The region between 66 and 76 base pairs before the cap site contains a variant of the canonical sequence G-G-C-T-C-A-A-T-C-T found preceding many other genes at a similar location, and this region may modulate the efficiency of transcription. The sequence of 425 nucleotides preceding the rabbit beta-globin gene is reported.