To determine whether 1 alpha, 25-dihydroxyvitamin D3-dependent increases in intestinal calcium uptake require de novo protein and RNA synthesis, the effects of several inhibitors of these processes have been re-examined in vitro using cultured embryonic chick duodenum. To minimize the contributions of antibiotic toxicity to the interpretation of results, care was taken to examine inhibitor effects at early times after the onset of the 1 alpha, 25-dihydroxyvitamin D3 response. Cycloheximide at a concentration of 5 microM blocked hormone-dependent calcium uptake at all times examined (6 to 24 h). Actinomycin D was similarly effective at 6 to 12 h. The effects of cycloheximide were totally reversible while actinomycin D inhibition was only partially reversible. These compounds inhibited protein or RNA synthesis by 68.4 +/- 1.4 and 51.4 +/- 1.1%, respectively. Anisomycin, another inhibitor of polypeptide chain elongation and alpha-amanitin, an inhibitor of RNA polymerase I, also blocked 1 alpha, 25-dihydroxyvitamin D3-dependent calcium uptake after 12 h in culture. These results further strengthen the hypothesis that 1 alpha, 25-dihydroxyvitamin D3 stimulates intestinal calcium transport via a nuclear mechanism involving new gene expression.