We have studied the transient expression of a cloned rabbit hemoglobin beta 1 gene after its introduction into HeLa cells. Two and one-half days after transfection using the calcium phosphate technique we extracted RNA from the entire cell population and analyzed it by the S1 nuclease hybridization assay. Transcripts were barely detectable when beta-globin gene-plasmid recombinants were used. However, 200 times more beta-globin gene transcripts were found when the beta-globin gene recombinants also contained SV40 DNA, and 90% of these transcripts (about 1000 per cell) had the same 5' end as authentic rabbit globin mRNA. In the latter case, abundant production of beta-globin protein was readily detected in a fraction of transfected cells by immunofluorescent staining. Enhancement of globin gene expression was dependent on SV40 sequences acting in cis, but independent of the viral origin of DNA replication. The enhancing activity was associated with the 72 bp repeated sequence element located at the beginning of the viral late gene region. Viral DNA fragments containing the transcriptional enhancer element could act in either orientation at many positions, including 1400 bp upstream or 3300 bp downstream from the transcription initiation site of the rabbit beta-globin gene. These studies define a class of DNA elements with a mode of action that has not been heretofore described. The activation of genes by specific enhancer elements seems to be a widespread mechanism that may be used for the regulation of gene expression.