AbstractFreeze‐thaw damage to thylakoids in spinach leaves has been simulatedin vitro, using a complex, defined artificial stroma medium. The resulting mechanical damage was quantified by measuring the loss of the marker protein plastocyanin from the thylakoid lumen, which is released as a result of membrane rupture. Loss of plastocyanin was already apparent at 0°C and became more severe at subzero temperatures. The time course of plastocyanin loss during freezing was biphasic: after an initial rapid loss, plastocyanin release was linearly dependent on incubation time. In short‐term experiments a linear dependence on freezing temperature was observed. Solute diffusion into the thylakoids, leading to influx of water and eventually membrane rupture, has been observedin vitroas well as after freezing of le