The conversion of 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) takes place mainly in the kidney and is catalyzed by the enzyme 1α-hydroxylase. Parathyroid hormone (PTH) and 1,25-(OH)₂D₃ are well-known regulators of this tightly controlled step, but the mechanisms by which they modulate 1α-hydroxylase activity have not been fully delineated. Northern analysis showed PTH and forskolin rapidly and transiently increase 1α-hydroxylase expression in AOK-B50 cells and HKC-8 cells. Actinomycin D treatment blocks the increase, but cycloheximide does not. No decrease of 1α-hydroxylase transcript by 1,25-(OH)₂D₃ was observed in either cell line, although 24-hydroxylase levels were strongly induced by 1,25-(OH)₂D₃ treatment. 1,25-(OH)₂D₃ suppressed the 1α-hydroxylase transcript in vivo both in the presence and absence of exogenously supplied PTH. These results suggest that the stimulatory action of PTH is directly on the 1α-hydroxylase gene, while the repressive action of 1,25-(OH)₂D₃ does not involve the parathyroid gland but is nevertheless indirect.