A sandwich-type enzyme immunoassay for human 28 kDa vitamin D-dependent calcium binding protein (calbindin-D) was established with a sensitivity of 1 pg/tube. Antisera were generated in rabbits injected with highly purified human kidney calbindin-D, and specific antibodies to calbindin-D were purified by the use of a column of calbindin-D-coupled Sepharose. The purified antibodies showed a single band at the position corresponding to calbindin-D on an immunoblotting test with a crude extract of human kidney. The assay system consisted of polystyrene balls with immobilized F(ab')2 antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to 28 kDa calbindin-D, showing no cross-reactivity with other calcium binding proteins such as S-100a0 (alpha alpha), S-100b (beta beta), parvalbumin and calmodulin. The assay was also reproducible (coefficients of variation between assays were less than 10%). With the present method, immunoreactive calbindin-D could be detected in various human tissues, with major concentrations in kidney and brain. The values for immunoreactive calbindin-D in various body fluids of healthy subjects varied from undetectable in serum and semen to 3.8 +/- 2.0 (SD) micrograms/g creatinine in urine and 2.9 +/- 0.8 (SD) micrograms/l in cerebrospinal fluid. Immunohistochemically, the calbindin-D in human kidney was localized in epithelial cells of distal tubules.