Based on the contents of the database S/MARt DB, the most comprehensive data collection of scaffold/matrix-attached regions (S/MARs) publicly available thus far, we initiated a systematic evaluation of the stored data. By analyzing the 245 S/MAR sequences presently described in this database, we found that the S/MARs contained in this collection are generally AT-rich, with certain significant exceptions. Comparative analyses showed that most of the AT-rich motifs which were found to be enriched in S/MARs are also enriched in randomized S/MAR sequences of the same AT content. Some sequence patterns previously suggested to be characteristic for S/MARs were also investigated, among them potential binding sites for homeodomain transcription factors. Even though hexanucleotides containing the core motif of homeodomain factors were frequently observed in S/MARs, only a few potential binding sites for these factors were found enriched when compared with regulatory regions or exon sequences. All our analyses indicated that, on average, the observed frequency of motifs in S/MAR elements is largely influenced by the AT content. Our results can serve as a guideline for further improvements in the definition of S/MARs, which are now believed to constitute the functional coordinate system for genomic regulatory regions.