Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)₂D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)₂D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)₂D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared to the holo CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)₂D but without a concomitant rise in 24-hydroxylase activity. However, over-expression of CYP24-SV in HD-11 and THP-1 cells, reduced synthesis of 1,25-(OH)₂D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)₂D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant-negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)₂D in human health and disease.