The transcriptional enhancer effect is used in many, if not all, organisms for remote control of gene transcription. An enhancer DNA can dramatically stimulate transcription of a linked gene from positions either 5' or 3' to the gene. Both the proximal promoter and the distal enhancer sequences are binding sites for transcription factors. Interaction between promoter and enhancer is mediated by these factors, presumably via looping out of the intervening DNA. Here we report that the extent of remote activation by an enhancer depends on characteristics of that intervening DNA. Using Beta-globin and SV40 T-antigen test genes, we show that the effect of an SV40 enhancer is transmitted to the responsive promoter, with little or no loss of efficiency, through certain segments of mammalian DNA derived from rabbit beta-globin or mouse alpha-globin gene regions. By contrast, a strong reduction of enhancer activity is observed with certain spacer segments of prokaryotic DNA (from plasmid pBR322 or phage lambda) or sequences of high (G + C) content from eukaryotic genes. We have analyzed more closely sequences that are more or less permissive for transmission of the transcriptional enhancer effect. It appears that these permissive sequences generally have a high (A + T) content and notably a very low abundance of CpG dinucleotides. By contrast, (G + C)-rich DNA segments with high local densities of CpG were the most deleterious for long-range enhancer action. We note that the latter sequence composition is typical for "CpG islands" of many mammalian genes, including housekeeping genes and the human alpha-globin gene. This may be related to the finding that promoters of most cell type-specific genes, whose activity depends on a strong enhancer, do not contain CpG islands. Most likely, the spacer DNAs of typical cell type-specific genes have evolved to allow maximal transmission of the enhancer effect.