Urinary N1-acetylspermidine (N1SPD) and N8-acetylspermidine (N8SPD) were measured in 24-hr urine specimens from 42 patients with colon adenocarcinoma and 29 healthy controls to assess their use as markers for colon cancer screening. Serial spot urines in four controls demonstrated significant fluctuations in these polyamine levels throughout the day without a distinct circadian pattern and therefore all subsequent analyses were performed on 24-hr collections. Both N1SPD and N8SPD were significantly increased in colon cancer patients compared to controls. Neither test correlated with tumor stage or location, but N8SPD was elevated in patients with poorly differentiated adenocarcinoma when compared to moderate or well-differentiated tumors. Using receiver operator characteristic (ROC) analysis, N1SPD had a higher information content than N8SPD, N1SPD + N8SPD, or the ratio of N1SPD/N8SPD and at a normal cut-off value of 4.0 nmol/mg creatinine, yielded a 95% specificity and 50% sensitivity for colon cancer.