To understand the mechanisms underlying increased expression of Myc protein in human urinary bladder cancer, expression of c-myc mRNA and the copy number of the c-myc gene were determined. Expression of mRNA was measured by quantitative RT-PCR in 40 urothelial carcinomas and in 18 histologically normal mucosae. Mean expression in tumors was significantly increased (3.23+/-2.63 AU vs. 1.90+/-0.95 AU, p < 0.023) and exceeded the highest level in normal mucosa in 15 (37.5%) tumors. The c-myc gene copy number was higher than in leukocytes and normal bladder mucosa in 14 of 40 tumors, but only 3 among these showed a more than 4-fold increase indicative of gene amplification. Most, but not all, tumors with elevated expression displayed an increased gene copy number (p < 0.0001). In line with other studies of the protein level, no significant association either of c-myc mRNA over-expression or of increased gene copy number with tumor stage or grade was observed. The data indicate that elevated mRNA expression as a consequence of increases in c-myc gene copy number often underlies Myc protein over-expression in bladder cancer. This increase may be a consequence of, most frequently, chromosome 8q gain and, occasionally, gene amplification, while in some tumors deregulation of mRNA expression occurs without evident changes in the c-myc gene copy number.