High- and low-energy collision experiments, by means of double-focusing and ion-trap mass spectrometers, respectively, performed on the M+ and [M-H2O]+. ions of 7-alpha- and 7-beta-hydroxycholesterol lead to a clear structural characterization of the two stereoisomers. In particular, the beta isomer shows peculiar fragmentation pathways due to interaction between the hydroxyl group in position 7-beta and the hydrogen atom on C(15).