Abstract When the human β-globin gene is transfected into Hela cells, no β-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable β-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of β-globin promoter mutants indicates that between 75 and 128 bp of sequence 5′ to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, β-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5′-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate β-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.