The in vivo stimulation of vitamin D-dependent calcium-binding protein (9 K CaBP) synthesis by 1,25-dihydroxycholecalciferol [l,25(OH)₂D₃]in the rat duodenum has been analyzed using a specific [³²P] complementary DNA probe for rat 9 K CaBP and inhibitors of RNA transcription (actinomycin D, α-amanitin) or protein synthesis (cycloheximide). The relative amounts of 9 K CaBP messenger RNA (mRNA) were assayed by dot-blot hybridization and the relative amounts of 9 K CaBP by RIA. Both inhibitors were injected at doses which significantly inhibited by 80–95% [³⁵S]methionine or [³H]uridine incorporation into protein and RNA, respectively. In vitamin Ddeficient rats, a single 1,25(OH)₂D₃ injection (650 pmol/100 g BW) resulted in a rapid production of 9 K CaBP mRNA which was significantly detectable as early as 3 h, and was followed by an increase of 9 K CaBP levels. Injection of actinomycin D (25 μg/100 g BW) 1 h before 1,25(OH)₂D₃ treatment and repeated every 4 h did not prevent the hormone-induced elevation of duodenal CaBP mRNA, even when the actinomycin dose was doubled and given 2 h before hormonal treatment. a-Amanitin (2 μg/100 g BW) also failed to block the hormonal stimulation. The protein synthesis inhibitor cycloheximide (25 μg/100 g BW) did not cause any change in the l,25(OH)₂D₃-induced CaBP mRNA but blocked the CaBP increase after hormone injection. Thus, transcription inhibitors did not prevent the in vivo hormone-induced elevation of 9 K CaBP mRNA, which suggests that 1,25(OH)₂D₃ increases 9 K CaBP synthesis by increasing 9 K CaBP gene expression at one or more posttranscriptional steps. More precise data will be obtained by measuring the rate of 9 K CaBP gene transcription on isolated nuclei from rat duodenum. (Endocrinology119: 2476–2483, 1986)