The present investigation was conducted to determine a single dose of the carcinogen N-methyl-N-nitrosourea (MNU) which remains subcarcinogenic in a heterotopically transplanted rat urinary bladder with a communicating reservoir (HTB) either in a urine-free or urine-existing environment. A single dose of MNU, either 0.1 mg or 0.025 mg, was instilled directly into the HTB, and was followed by weekly instillation of normal rat urine or 2.1% NaCl solution (equiosmolar to the urine) for up to 52 weeks. Bladder tumors observed were divided into 2 categories depending upon their location. Those arising at the hyperplastic foci which were induced by mechanical irritation by the connector tip were referred to as inflammatory polyp (IP)-related tumors and those arising in the region free from the IP changes were referred to as IP-unrelated tumors. The results indicated that both carcinogen levels fail to induce IP-related or IP-unrelated tumors in the urine-free environment. In the presence of urine, however, the high MNU dose (0.1 mg) was tumorigenic at both IP-related and IP-unrelated sites, whereas, the low MNU dose exhibited tumorigenicity only at the IP-related site. Physical irritation by the connector enhanced MNU-initiated tumorigenesis at the connector tip only in the presence of urine in bladder lumen. Physical irritation per se induced tumors at the connector tip even without prior carcinogen treatment provided urine was present in the HTBs.