Intramolecular diffusion plays a role in protein folding as shown by kinetic experiments on two alpha-spectrin SH3 domain circular permutants (S19-P20s and N47-D48s), with different poly-glycine loop lengths. Insertion of up to 10 Gly residues does not alter the structure of the folded state nor the overall characteristics of the denatured ensemble. The apparent level of the energy barrier between the denatured and folded species increases linearly with the number of inserted glycines. This suggests that the transition state itself and/or possibly previous transient unstable intermediates are accessed with more difficulty when loop length is increased. The fact that the induced impediment is directly proportional to the number of Gly residues and not to the free energy difference in the folded state indicates that diffusion of different parts of the molecule relative to each other is taking place on going from the denatured ensemble to the transition state. Our results also suggest that transition state ensembles could be more homogenous than recently postulated.