The total amount and reactivity of SH-groups were determined in isolated duodenal brush-border membranes from rachitic chicks given 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3) before isolation of the membranes. 1,25(OH)2D3 treatment significantly increased the total amount of SH groups (9.7 +/- 2.3 vs. 23.9 +/- 2.1, P less than 0.001, n = 6) in brush-border membranes solubilized in 1% sodium dodecyl sulfate. The rate of reaction (reactivity) of membrane-bound SH-groups, determined with the fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), was also significantly enhanced by the intravenous injection of various doses (0.005-0.10 microgram) of 1,25(OH)2D3 into vitamin D-deficient chicks. An increase in reactivity occurred as early as 10 min after dosing of the chicks with 1,25(OH)2D3. Fluorescence scanning of the membrane proteins labeled with DACM and separated by polyacrylamide gel electrophoresis revealed three major peaks of fluorescence and a generally higher degree of fluorescent labeling of these and many other proteins in the membranes isolated from the 1,25(OH)2D3-treated chicks. The physiological significance of the 1,25(OH)2D3-mediated increase in the reactivity and total amount of membrane-bound SH-groups in terms of vitamin-D-dependent epithelial transport and epithelial membrane properties needs to be further explored.