We have developed a rapid and simple method for studying the transcription of cloned eucaryotic genes, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors). Because COS cells produce SV40 T antigen and are permissive for SV40 viral replication, transfected SV-ORI plasmids replicate to a high copy number. SV-ORI plasmids carrying a human alpha-globin gene are also replicated in COS cells. Moreover, the alpha-globin gene is faithfully transcribed to produce high levels of RNA, which is accurately processed to produce authentic alpha-globin mRNA. We have used this transcription system to demonstrate that a sequence located between 55 and 87 base pairs upstream from the mRNA capping site is required for efficient transcription of the alpha-globin gene in COS cells.