The relationship between temperature-dependent changes in phagocytotic activity of Acanthamoeba castellanii and the fatty acid composition and physical properties of plasma membrane-enriched fractions were determined in cultures acclimated to 30 degrees C and 15 degrees C. Chilling (from 30 degrees C to 15 degrees C) had a very pronounced short-term inhibitory effect on phagocytosis only in stationary-phase cultures, which displayed a low degree of fatty acid unsaturation. A subsequent increase in phagocytosis by these cells was correlated with a low-temperature-induced increase in fatty acid unsaturation (shown previously [Jones, Lloyd and Harwood (1993) Biochem. J. 296, 183-188] to be due to n-6 desaturase induction). Plasma membrane-enriched fractions from 15 degrees C-acclimated cells also showed a marked increase in the relative proportion of polyunsaturated fatty acids. Steady-state fluorescence depolarization studies, using the membrane probe diphenylhexatriene, revealed increases in plasma membrane order with decreasing assay temperature. Over the upper assay-temperature range (25-40 degrees C), fluorescence anisotropy values were higher in membranes from 30 degrees C-acclimated cells; a 3.3 degrees C relative displacement of plots indicated that temperature-induced changes in membrane lipid composition compensated for approx. 22% of the ordering effect of low temperature. Changes in the temperature-dependence of fluorescence anisotropy, possibly corresponding to lateral phase separations or alterations in other bulk physical properties of membranes, occurred between 20 and 25 degrees C in membranes from 30 degrees C-acclimated cells and between 15 and 20 degrees C in membranes from 15 degrees C-acclimated cells. Fluorescence anisotropy plots were superimposed at assay temperatures between 5 and 15 degrees C. Short-term phagocytotic rates in whole cells decreased with assay temperature. Arrhenius discontinuities in rates of phagocytosis occurred at approx. 25.0 degrees C and 17.5 degrees C in 30 degrees C- and 15 degrees C-acclimated cells respectively, and in each case were thus within the temperature ranges of slope-change in the corresponding fluorescence anisotropy plots. The results show a direct correlation between plasma membrane fatty acid unsaturation, membrane physical properties and phagocytotic activity in A. castellanii. Therefore, a specific integrated physiological process has been correlated with fatty acid desaturase induction for the first time.