It is well established that changes in the phenotype depend much more on changes in gene expression than on changes in protein-coding genes, and that cis-regulatory sequences and chromatin structure are two major factors influencing gene expression. Here, we investigated these factors at the genome-wide level by focusing on the trinucleotide patterns in the 0.1- to 25-kb regions flanking the human genes that are present in the GC-poorest L1 and GC-richest H3 isochore families, the other families exhibiting intermediate patterns. We could show 1) that the trinucleotide patterns of the 25-kb gene-flanking regions are representative of the very different patterns already reported for the whole isochores from the L1 and H3 families and, expectedly, identical in upstream and downstream locations; 2) that the patterns of the 0.1- to 0.5-kb regions in the L1 and H3 isochores are remarkably more divergent and more specific when compared with those of the 25-kb regions, as well as different in the upstream and downstream locations; and 3) that these patterns fade into the 25-kb patterns around 5kb in both upstream and downstream locations. The 25-kb findings indicate differences in nucleosome positioning and density in different isochore families, those of the 0.1- to 0.5-kb sequences indicate differences in the transcription factors that bind upstream and downstream of genes. These results indicate differences in the regulation of genes located in different isochore families, a point of functional and evolutionary relevance.