We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.