Ornithine decarboxylase (ODC) and polyamines (putrescine, spermidine, and spermine) are crucial for cell proliferation. Recently, elevated ODC activity and polyamine levels have been suggested as biological markers for human colon cancer. In this study, we measured ODC activity and the levels of polyamines (putrescine, spermidine, spermine, and cadaverine) and acetyl-putrescine in human colonocytes isolated from cancerous areas compared to the adjacent normal colon tissue. In addition, ODC mRNA expression was compared between both groups. We found that colonocytes isolated from cancerous areas had significantly higher mean value of ODC activity, putrescine, spermidine, spermine, and cadaverine levels up to 1480%, 470%, 260%, 380%, and 510% respectively compared to colonocytes isolated from the adjacent normal colonic mucosa. No difference was found in acetyl-putrescine levels between cancerous and normal colonocytes. Steady-state levels of ODC mRNA were slightly elevated in cancerous colonocytes relative to normal colonocytes in two of three paired samples. However, the increase in ODC mRNA levels is not sufficient to account for the increase in ODC activity suggesting that colonocyte ODC activity is regulated post-transcriptionally.