To obtain a group-specific antibody to 1α,25-dihydroxyvitamin D and its derivatives having the 1α,3β-dihydroxylated A-ring structure, 1α-hydroxy-25,26,27-trinorvitamin D 3-24-oic acid was synthesized as a haptenic derivative by a new convenient synthetis route. Three kinds of polyclonal antibodies were elicited in rabbits against the hapten conjugated with bovine serum albumin, and their specificity was examined by cross-reactivities in a radioimmunoassay procedure. One of the antibodies was found to show a suitable binding property by addition of a small amount of the plasma from vitamin D-deficient rats. Dose-response curves with measurable ranges applicable to biological specimens were obtained for 1α,25-dihydroxyvitamin D 3, 1α,25-dihydroxyvitamine D 2, (24R)-1α,24,25-trihydroxyvitamin D 3, (23S,25R)-1α,25-dihydroxyvitamin D 3 26,23-lactone, and 22-oxacalcitriol by the radioimmunoassay system using this improved antibody. The antibody will be widely useful for developing profile analyses of 1α,25-dihydroxyvitamin D and its derivatives (metabolites or synthetic analogs) having the 1α,3β-dihydroxylated A-ring, as well as immunoassays measuring each of these secosterols individually. (Steroids 59:404-411, 1994)