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OPTICAL activity manifested by chiral molecules and crystals is of long-standing interest in physics, chemistry, biology and geology^1,2. The structure of chiral lattices in two and three dimensions may provide insights into chiral discrimination, and chiral phases play an important part in the physics and applications of liquid-crystal and amphiphilic films^3–7. Here we report the observation of spontaneous separation of chiral phases in an oriented monolayer of rigid, chiral amphiphiles deposited on mica. Atomic force microscopy of the ordered films reveals domains of mirror-image structures. We propose that this may be the signature of separation into domains of pure enantiomers, although other possibilities exist. This separation of chiral phases in two dimensions may be considered analogous to the spontaneous resolution of enantiomers in three-dimensional crystallization, as exploited by Pasteur in 1848 to isolate …