Cytogenetic studies of rat neurogenic tumor lines induced by ethylnitrosourea (ENU) have shown specific involvement of chromosome 4. The study reported here further characterizes the association of chromosome 4 abnormalities in rat tumor cell lines with regard to etiological agent, tissue of origin, and tumorigenic potential of cloned lines. Lines from rat gliomas induced by avian sarcoma virus did not show abnormalities of chromosome 4. ENU-induced rat tumors of nonneurogenic origin had numerical and/or structural abnormalities of chromosome 4 in seven of nine cell lines. In a comparison of two tumorigenic and two nontumorigenic cloned cell types from the same ENU-induced rat neural tumor, all showed excess chromosome 4. In addition, preneoplastic nervous system tissue, exposed to ENU in vivo, was cultured and sequentially monitored for the concurrent development of chromosome abnormalities and neoplastic properties. Abnormalities of chromosome 4 were observed in 3 of 14 tumorigenic lines and one nontumorigenic clone. The remaining lines had normal karyotypes or abnormalities involving chromosomes other than chromosome 4. Our results suggest that chromosome 4 abnormalities appear late in tumor development, are probably secondary to the tumorigenic potential of the studied cell lines, and apparently are not tissue specific. However, abnormalities of chromosome 4 may be associated preferentially with ENU oncogenesis.