When Tetrahymena pyriformis cells grown at 39°C were chilled to 15°C, a rapid desaturation of microsomal phospholipid-bound fatty acids was observed. A concurrent but even more rapid change in the physical properties of the microsomal lipids was detected by steady-state fluorescence polarization measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in lipid multilamellar vesicles. Whereas polarization vs. temperature plots of lipids from 39°C-grown cells showed discrete break points (abrupt slope changes thought to indicate altered phase separation rates) at characteristic temperatures, plots made using lipids from equivalent cells chilled to 15°C for 15 or 20 min lacked such clearly defined break points. The sharp break points reappered in plots of microsomal lipids from cells maintained at 15°C for 30 min or longer, but in these curves the temperature of each break point was several degrees lower than in 39°C-cell lipids and nearly the same as in cells fully acclimated to low temperature (48 h or more at 15°C). Fluorescence polarization studies on mixtures of natural lipids from 39°C-cells and cells shifted to 15°C or on mixtures of natural and synthetic lipids revealed that each of the two break points in a polarization vs. temperature plot can respond to changes in lipid composition independently of the other. It is concluded that the expeditious desaturation of certain key fatty acids, perhaps coupled with limited retailoring of phospholipid molecular species, leads to pronounced physical changes in Tetrahymena microsomal membranes as the first step of low temperature acclimation.