Abstract Proliferation of smooth muscle cells (SMC), enhancement of polyamine biosynthesis and increase in polyamine level in response to deendothelialization in the rat aorta were studied. [ 3H]Thymidine incorporation into SMC in aortas denuded with a balloon catheter began 25 h after injury, and maximal incorporation occurred 33–37 h after injury. Afterwards, [ 3H]thymidine incorporation declined, approaching the baseline level, but was slightly higher than that of sham-operated controls until 14 days after injury. Intimal thickening started 7 days after injury, and peaked at 21 days. Prior to these proliferative changes in aortic SMC, a rapid and transient increase in ornithine decarboxylase (ODC) activity was observed within 8 h after injury. There was no significant difference in ODC activity between injured and intact aortas after 4 days. The levels of polyamines, putrescine, spermidine, and spermine increased and were maximal at 48 h after injury, 8.1, 3.4 and 1.4 times the control levels, respectively. Increased levels of polyamines, in particular spermidine, continued until 7 days after injury. These results suggest that the enhancement of polyamine synthesis and the increased polyamine content of the aorta play important roles in the proliferation of SMC and in the development of intimal thickening, particularly in the initial proliferative response of medial SMC after deendothelialization.