We describe the construction and characterization of miniplasmid vectors that can be used to isolate and express normal and mutant alleles of the human beta-globin gene. These vectors, designated pi SV beta plasmids, contain a bacterial origin of replication and selectable marker, a 5'-flanking beta-globin DNA fragment that can be used for recombination screening (Seed, 1983), and simian virus 40 (SV40) sequences that allow accurate and efficient expression of the beta-globin gene transfected into mammalian cells. We show that pi SV beta plasmids can be used to select cloned beta-globin genes from a bacteriophage lambda library of genomic DNA, and that plasmids containing the beta-globin gene linked to the SV40 enhancer sequence can be excised from the phage, circularized and recovered by transformation of Escherichia coli. Analysis of the beta-globin transcripts produced by the recovered pi SV beta recombinant plasmids after transfection into COS cells and replication to high copy number, indicates that the beta-globin gene is accurately transcribed, but a substantial fraction of the transcripts are the result of readthrough from sites within the vector. In contrast, when these plasmids are transferred into HeLa cells beta-globin RNA is accurately initiated and little readthrough transcription is observed. These results indicate that HeLa cells are more suitable than COS cells for studying mutant beta-globin genes, even though the copy number of the pi SV beta plasmids is much higher in COS cells.