Polyamines are short-chain aliphatic amines required for normal cellular growth that are ubiquitously found in all living tissues. Polyamine content has been shown to correlate with cellular proliferation. Quantitation of polyamines may thus provide a biochemical measure of proliferation in the colorectal mucosa where dysregulated epithelial proliferation is associated with colorectal cancer risk. A case-control study was conducted to validate the hypothesized association between mucosal polyamine measurements and colorectal cancer risk. Polyamines were measured in 4-6 multiple rectal mucosal biopsies from 11 normal control subjects and seven case patients with colon cancer. Compared with the controls, mean polyamine measurements, after adjustment for age and sex, were significantly increased for spermidine (P < 0.003) and spermine (P < 0.017). Subsequent analysis indicated that in controls 1-4 biopsies appeared adequate to characterize an individual. However, mucosal polyamines in the cases exhibited more sampling variability, requiring 4-8 biopsies to achieve an acceptable level of reliability. After adjustment for age and sex, the odds ratios for spermidine and spermine levels, compared to the controls, were 4.8 (95% confidence interval: 1.6-33.7) and 2.3 (1.2-6.3), respectively. The results of this study indicate that increases of mucosal polyamine measurements, after taking the sampling and methodological variability into account, are significantly associated with colorectal cancer risk, and suggest that polyamine measurements in rectal mucosa may play an important role as biomarkers for identifying high-risk individuals and/or for using as intermediate endpoints in prevention trials.