Inhibitors of RNA synthesis, actinomycin D and cordycepin, stimulate chick duodenal alkaline phosphate [alk Pase] activity by means which are additive to the stimulation by the biologically active metabolite of vitamin D3, 1,25(OH)2D3, and its analogue, 1.alpha.OH D3. The protein synthesis inhibitor, cycloheximide, inhibited basal alk Pase activity and blocked its stimulation by actinomycin D and 1,25(OH)2D3. Both actinomycin D and cycloheximdie blocked the ability of 1,25(OH)2D3 and 1.alpha.OHD3 to raise serum Ca levels. The paradoxical stimulation of duodenal alk Pase activity by RNA synthesis inhibitors additive to the stimulation by 1,25(OH)2D3 suggests that alk Pase activity is controlled by mechanisms other than gene activation.