VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.