The folding kinetics of G-quadruplex forming sequences is critical to their capacity to influence biological function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes containing only two G-quartets formed from sequences r[(GGA)(3)GG] and r[(GGUUA)(3)GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K+, irrespective of the number of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiological [K+] (90 mM) (tau = similar to 60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (tau = similar to 700 ms), and we found that the time required to fold a UUA looped variant (tau > 100 s, 500 mM K+) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biology.