Previously, it has been reported that human telomeric DNA sequences could adopt in different experimental conditions four different intramolecular G-quadruplexes each involving three G-tetrad layers, namely Na + -solution antiparallel-stranded basket form, K + -crystal parallel-stranded propeller form, K + -solution (3+1) Form 1 and K + -solution (3+1) Form 2. Here we present a new intramolecular G-quadruplex adopted by a four-repeat human telomeric sequence in K + solution (Form 3). This structure is a basket-type G-quadruplex with only two G-tetrad layers: loops are successively edgewise, diagonal and edgewise; glycosidic conformations of guanines are syn•syn•anti•anti around each tetrad; each strand of the core has both a parallel and an antiparallel adjacent strands; there are one narrow, one wide and two medium grooves. Despite the presence of only two G-tetrads in the core, this structure is more stable than the three-G-tetrad intramolecular G-quadruplexes previously observed for human telomeric sequences in K + solution. Detailed structural elucidation of Form 3 revealed extensive base pairing and stacking in the loops capping both ends of the G-tetrad core, which might explain the high stability of the structure. This novel structure highlights the conformational heterogeneity of human telomeric DNA. It revealed a new folding principle for Gquadruplexes and suggests new loop sequences and structures for targeting in human telomeric DNA.