Nucleic acid structure-function correlations are pivotal to major biological events like transcription, replication, and recombination. Depending on intracellular conditions in vivo and buffer composition in vitro, DNA appears capable of inexhaustible structure variation. At moderately acidic, or even neutral pH, DNA strands that are rich in cytosine bases can associate both inter- and intramolecularly to form i-tetraplexes. The hemiprotonated cytosine(+)-cytosine base pair constitutes the building block for the formation of i-tetraplexes, and motifs for their formation are frequent in vertebrate genomes. A major control element upstream of the human c-myc gene, which has been shown to interact sequence specifically with several transcription factors, becomes hypersensitive to nucleases upon c-myc expression. The control element is asymmetric inasmuch as that one strand is uncommonly rich in cytosines and exhibits multiple motifs for the formation of i-tetraplexes. To investigate the propensity for their formation we employ circular dichroism (CD) in combination with ultra violet (UV) spectroscopy and native gel electrophoresis. Our results demonstrate the cooperative formation of well-defined i-tetraplex structures. We conclude that i-tetraplex formation occurs in the promoter region of the human c-myc gene in vitro, and discuss implications of possible biological roles for i-tetraplex structures in vivo. Hypothetical formation of intramolecular fold-back i-tetraplexes is important to c-myc transcription, whereas chromosomal translocation events might involve the formation of bimolecular i-tetraplex structures.