Polyamines (putrescine, spermidine, and spermine) are synthesized primarily from ornithine via ornithine decarboxylase (ODC) in mammals. Although avian tissues contain ODC activity, little is known about intracellular sources of ornithine for their polyamine synthesis. This study tested the hypothesis that arginase and proline oxidase contribute to polyamine synthesis in chickens. Kidney, jejunum, leg muscle, and liver from 0-, 7-, 14-and 21-day-old broiler chickens were assayed for the activities of arginase, proline oxidase (POX), ornithine aminotransferase (OAT), and ornithine decarboxylase (ODC). Kidney slices were also used to determine 14 C-polyamine synthesis from [U-14 C]arginine and [U-14 C]proline. Furthermore, these tissues and plasma were analyzed for polyamines. Results indicate that all tissues contained OAT (mitochondrial) and ODC (cytosolic) activities, but arginase and POX activities were only detected in the mitochondria of chicken kidneys. Renal POX and arginase activities were greater at 7 days of age compared to newly hatched birds, and declined by Day 14. Renal arginase activity was greater at 21 days compared to 14 days of age, but there was no change in renal POX activity during that same period. Concentrations of polyamines in the kidneys and plasma were greater on Day 7 compared to Day 0 and decreased thereafter on Days 14 and 21. Kidney slices readily converted arginine and proline into polyamines, with peak rates being on Day 7. Concentrations of putrescine, spermidine and spermine in the plasma of chickens were about 20-to 100-fold greater than those in mammals. Our results indicate that polyamines are synthesized from arginine and proline in avian kidneys. Unlike mammals, polyamines released from the kidneys are likely the major source of polyamines in the blood and other extra-renal tissues in chickens.