Data from experiments addressing the putative biological function of 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] remain controversial. We have begun to study the role of 24,25(OH) 2 D 3 using the powerful technique of gene inactivation by homologous recombination in embryonic stem (ES) cells. A null mutation in the 25-hydroxyvitamin D 24-hydroxylase (24-OHase) gene has been engineered in ES cells. Targeted ES cells were injected into mouse blastocysts and one of the resulting chimeric mice has transmitted the targeted allele to its progeny. Animals that are heterozygous for the engineered mutation are normal and fertile. Animals homozygous for the targeted 24-OHase mutation are born with the expected Mendelian frequency of 25% (36/154); however, about one-half of the homozygotes died within one week after birth. We suspect that the incomplete penetrance of the homozygous phenotype may be due to the mixed genetic background (129Sv C57 B1 6) of the animals. Preliminary data suggest a mild hypercalcemia in homozygous mutants; histological examination revealed normal bone structure. Homozygous animals that survive are fertile. Interestingly, preliminary results suggest that bone development was abnormal in homozygotes born of homozygous females. These analyses should yield valuable insight into the biological role of 24,25(OH) 2 D 3 in mineral homeostasis and bone development.