The electron spin-echo envelope modulation (ESEEM) technique was used to investigate the formation mechanism of the mesoporous material MCM-41. The spin-probes 4-(N, N-dimethyl-N-hexadecyl)ammonium-2, 2, 6, 6, -tetramethyl piperidine-oxyl iodide (CAT16) and 5-doxyl stearic acid (5DSA) were introduced into the surfactant (cetyltrimethylammonium bromide, CTAB) solution in minute amounts followed by the addition of a base and a silica source to initiate the reaction. The reaction was then quenched at different times by rapid insertion into liquid nitrogen. The preservation of the micellar structure upon freezing was proved by a series of ESEEM measurements carried out on 5DSA in CTAB solutions of various concentrations, which showed that the 14N modulation depth was sensitive to the transition from spherical to cylindrical micelles. Variations in the immediate environment of the spin-probes occurring during the room temperature formation of MCM-41 were followed by tracing the 2H modulation depth k(2H) induced by α-d2-CTAB molecules and D2O. For both spin-probes, k(2H) of α-d2-CTAB decreased throughout the reaction, whereas k(2H) of D2O showed a small increase. In all cases, the time evolution of k(2H) revealed two stages: one that lasted for the first ∼12 min, during which most changes have occurred, followed by a second, longer one with mild changes. The reduction of k(2H) of α-d2-CTAB in the case of 5DSA was assigned to its displacement toward the organic core, driven by charge repulsion between negatively charged silicate oligomers at the interface and the negative polar head of 5DSA. Considering the different position of the nitroxide spin label in CAT16 and 5DSA with respect to the α-position in the CTAB molecules, the decrease in k(2H) for CAT16 was attributed to an upward displacement, and a protrusion into the soft silica layer, driven by steric consideration and charge attraction. The slight increase in k(2H) due to D2O shows that the silica layer formed in the room temperature synthesis is water rich such that the density of water and OH groups in the vicinity of the spin-probes increases. The majority of the water, however, is easily removed just by filtering the solid formed and drying at room temperature. Finally, evidence for the rearrangement of surfactant molecules and the increase of the aggregate size during the first stage of the reaction was obtained from changes in the echo decay time.