We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 into vitamin D-deficient chicks produces a marked accumulation of putrescine in the duodenum by an interconversion pathway. In the present study, we examined the effect of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine, a specific irreversible inhibitor of polyamine oxidase, on the duodenal putrescine synthesis induced by 1 alpha,25-dihydroxyvitamin D3. Addition of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to an assay mixture completely inhibited the activity of duodenal polyamine oxidase in vitro. Prior administration of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to chicks completely blocked the 1 alpha,25-dihydroxyvitamin D3-induced increase in duodenal accumulation of putrescine in vivo. The increase of the duodenal accumulation of putrescine by 1 alpha,25-dihydroxyvitamin D3 in vitamin D-deficient chicks coincided quantitatively with the amount of N1-acetylspermidine synthesized from spermidine after the injection of the vitamin into the chicks pretreated with the inhibitor of polyamine oxidase. These results clearly indicate that spermidine N1-acetyltransferase plays a preferential role in the increase in duodenal putrescine synthesis by 1 alpha,25-dihydroxyvitamin D3. The rapidly proliferating and maturing epithelium of small intestines will provide a good model for investigating the role of the interconversion of polyamine metabolism in cell growth and differentiation.