To evaluate whether membrane fluidity plays a role in regulating calcium flux across the intestinal brush border, we purified brush border membranes from different regions of the chick intestine and determined the relationship of their ability to transport calcium and their fluidity parameters, as determined by diphenylhexatriene fluorescence polarization. Raising the temperature from 4 to 37 C resulted in a 3-fold increase in calcium accumulation by duodenal brush border membrane vesicles (BBMV; 2.85 to 8.70 nmol/mg protein X 12 min). This rise in temperature was associated with a decrease in degree of polarization from 0.395 to 0.290. The methyl esters of cis- and trans-vaccenic acid markedly increased calcium uptake at all temperatures studied (4, 25, and 37 C); the trans isomer was more effective. Both cis- and trans-vaccenic acid lowered the degree of polarization of the BBMV from 0.340 to 0.311 and 0.293, respectively, at 25 C. cis- and trans-vaccenic acid were effective whether BBMV were prepared from vitamin D-deficient chicks or their hatchmates given 1,25-dihydroxyvitamin D [1,25-(OH)2D]. The ability to accumulate calcium correlated to the degree of polarization when calcium uptake by and degree of polarization of BBMV prepared from duodenum, jejunum, and ileum were compared at 25 C. The duodenal BBMV had the greatest calcium accumulation (9.4 nmol Ca/mg protein X 10 min) and the lowest degree of polarization (0.336); the ileal BBMV had the least calcium accumulation (3.5 nmol Ca/mg protein X 10 min) and the greatest degree of polarization (0.375); and the jejunal BBMV were intermediate (8.2 nmol Ca/mg protein X 10 min; 0.344). This rank order was the same whether the intestine was from vitamin D-deficient chicks or their hatchmates given 1,25-(OH)2D before they were killed. 1,25-(OH)2D stimulated calcium uptake by duodenal and jejunal BBMV, reaching a maximal effect at 4 h, but no changes in degree of polarization were observed during this period. A plot of the degree of polarization as a function of the reciprocal of absolute temperature showed an inflection point at approximately 25 C. Neither the slope of the plot nor the point of the inflection was altered by 1,25-(OH)2D. We conclude that chick intestinal BBMV membrane fluidity and calcium uptake correlate in relation to the effects of temperature, changes in membrane lipids, and regional differences in the intestine. However, 1,25-(OH)2D stimulates calcium uptake by BBMV without a detectable change in membrane fluidity.