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Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing β-galactosidase (RAd35β-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-κB response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl β- d -galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate β−galactosidase expression in infected cells. After infection with RAd35β-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of β-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.