Although microsatellites with functional effects have been described, generally, these repeats are considered as "junk" DNA in the same way as other repetitive sequences. Our aim was to investigate if certain microsatellites can have a functional role as cis-regulatory elements. A database was created of all short tandem repeats, from 2 to 10 bases, located in the first 10-kb 5' of the transcription start sites of all annotated genes of the human genome. Of 114 microsatellites selected based on their size and location in the promoter, 51 were found to be polymorphic. Using electrophoretic mobility shift assay (EMSA), we studied five repetitive motifs and three displayed specific protein binding which were found in 12 of the polymorphic microsatellites. An interesting microsatellite is the CTC/GAG repeat which, as double-stranded (DS) DNA, bound specificity protein 1 (SP1) with high affinity, formed triplexes in vitro and displayed differences in SP1 binding and triplex formation capacity for repeats with distinct numbers of repeat units. Interestingly, the polypyrimidine strand of the repeat (CTC) bound other proteins such as polypyrimidine tract-binding protein 1 (PTBP1) as single-stranded (SS) DNA, and a model with two alternative DNA conformations is proposed for these repeats. Distinct protein binding to DS DNA was also observed for different numbers of AAACA and AAAAT repeats. Our results suggest that certain microsatellites may act as cis-regulatory elements, controlling gene expression through transcription factor binding and/or secondary DNA structure formation. Due to their high polymorphism and abundance, they might represent an important source of quantitative genetic variation.