We have cloned multiple copies of 72-base-pair (bp) repeat transcriptional enhancer element from simian virus 40 in plasmid vectors upstream from the bacterial chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Two copies of the 72-bp repeat provided efficient activation of gene expression. Increasing the number of linked 72-bp units to four substantially improved the activation of gene expression, but further addition of enhancers diminished the activation of gene expression proportionally to the number of enhancers added. Enhancer regions containing 10 or more copies of the 72-bp sequence were very inefficient in gene activation. Plasmids containing such expanded enhancer regions also competed less efficiently in vivo for trans-acting enhancer-binding factors. These effects are specific for the enhancer element and are not produced by reiterations of the 21-bp promoter element. Multiple enhancer units placed upstream do not interfere with the accuracy of mRNA initiation directed by the simian virus 40 early promoter in these plasmid constructs but do severely inhibit the initiation of replication at the neighboring simian virus 40 origin of replication that overlaps the early promoter region. These results are consistent with the hypothesis that the structural alterations induced in the DNA by a large number of copies of the enhancer are not favorable for the activation of a linked gene or for the binding of factors believed to mediate the enhancement effect.