The Prins condensation of isobutylene with formaldehyde over iron substituted MFI catalysts has been investigated. The catalysts have been characterized by various methods: TPR for substituted iron, TPD, FTIR, XPS and microcalorimetry for acidity measurements. The catalytic tests show that the medium to weak acid strength of the active sites favors the selectivity to isoprene. A mechanism based on the selective activation of formaldehyde over these centers was proposed to explain the selectivity to isoprene. GaZSM-5 was also tested as a catalyst for this condensation, but its catalytic selectivity is rather close to that of AlZSM-5. In addition, all catalysts demonstrated high activity for the cracking of 4,4-dimethyl-1,3-dioxane, a possible intermediate.