In the present study we have examined the regulation of the polyamine biosynthetic pathway in a cell line deficient in ornithine decarboxylase (ODC) activity. These cells were unable to grow unless polyamines were provided in their growth medium. Seeding the cells in the absence of polyamines rapidly resulted in a cellular depletion of putrescine and spermidine. Although the cells were devoid of ODC activity they were demonstrated to express an inactive ODC which was feedback regulated by polyamines in a normal manner. Cells seeded in the absence of polyamines exhibited a marked increase in ODC synthesis rate which was not correlated with an equal change in the ODC mRNA level. The synthesis of S-adenosylmethionine decarboxylase (AdoMetDC) was also increased in the cells seeded in the absence of polyamines. However, this increase was essentially explained by a change in the amount of AdoMetDC mRNA. The addition of putrescine to the growth medium appeared to stimulate the conversion of AdoMetDC proenzyme into its two subunits, indicating a physiological role of putrescine in the regulation of AdoMetDC expression.