Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (P < 0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.