Recently, a new geochemical temperature proxy, the TEX86, was introduced. This proxy is based on the number of cyclopentane moieties in the glycerol dialkyl glycerol tetraethers (GDGTs) of the membrane lipids of marine Crenarchaeota, which changes as a response to temperature. However, until now, only sediment data have been used to establish this proxy, and experimental work is missing. We performed mesocosm studies with marine Crenarchaeota incubated at temperatures ranging from 5 to 35¿C and salinities of 27 and 35? to test the validity of the TEX86 proxy. Growth of marine Crenarchaeota in these mesocosms was evident from the substantial increase in the concentration of marine Crenarchaeotal membrane lipids with amounts up to 3400 ng/L. With increasing temperature, an increase in the number of cyclopentane moieties in the crenarchaeotal membrane lipids was observed. Different salinities did not show any effect on the GDGT distribution. The TEX86 showed a significant linear correlation to incubation temperature: TEX86 = 0.015 ¿ T + 0.10 (r2 = 0.79). This equation has a similar slope to the correlation obtained from core tops but differs in the intersection (TEX86 = 0.015 ¿ T + 0.28, r2 = 0.92). This difference is mainly determined by the smaller amount of the regioisomer of crenarchaeol in the incubation series compared to core top samples. These incubation experiments indicates that water temperature is indeed the major controlling factor for the membrane distribution of marine Crenarchaeota and confirms that the TEX86 proxy depends on a physiological response to regulate membrane fluidity.