Cloned rabbit beta-globin genes, modified in vitro by restructuring or site-directed mutagenesis, were introduced into mouse 3T6 cells, and the resulting transcripts were analyzed by nuclease S1 mapping. The first 109 bp preceding the cap site sufficed for maximal beta-globin transcription. This segment contained three functionally important regions of the ATA box region; the CCAAT box region; and the -100 region. The latter consists of an imperfect tandemly repeated sequence of 14 bp and 15 bp, both copies of which are required for optimal promoter function. Each of three regions contains two or more nucleotide positions where the introduction of point mutations reduces transcription by at least a factor of 2.