Although many biochemical and structural studies have demonstrated that DNA sequences containing runs of adjacent guanines spontaneously fold into G-quadruplex DNA structures in vitro, only recently has evidence started to accumulate for their presence and function in vivo. Genome-wide analyses have revealed that functional genomic regions from highly divergent organisms are enriched in DNA sequences with G-quadruplex-forming potential, suggesting that G-quadruplexes could provide a nucleic-acid-based mechanism for regulating telomere maintenance, as well as transcription, replication and translation. Here, we review recent studies aimed at uncovering the in vivo presence and function of G-quadruplexes in genomes and RNA, with a particular focus on telomeric G-quadruplexes and how their formation and resolution is regulated to permit telomere synthesis.