To investigate the patterns of alkylating agent-induced tumour formation, 40 fish (Rivulus marmoratus) were exposed to N-methyl-N-nitrosourea (MNU) at 50 ppm in 10 mM Hepes-buffered synthetic seawater for 2 h. Tumour incidence 4 months after exposure was approximately 95%, and mainly papillary thyroid tumours were induced. For elucidating the molecular event in Rivulus papillary thyroid carcinogenesis, we first analysed for ras gene mutations based on the known ability of MNU to induce point mutations. The amplified R. Marmoratus Ha-and Ki-ras gene exon 1 and 2 regions were checked along with confirming the presence and expression patterns of the DNA repair gene O(6)-methylguanine alkyltransferase (O(6)-MT) and other oncogenes (c-src, c-fos, and c-myc). Ha- and Ki-ras genes from 38 tumour samples were tested for point mutations with direct sequencing but were not found to contain mutations. In this report, based on the lack of Ha- and Ki-ras gene mutations in papillary thyroid tumour induction in R. Marmoratus, we suggest that Ha- and Ki-ras gene-independent pathways such as ret/PTC rearrangements and other activations are involved in Rivulus papillary thyroid carcinogenesis. Teratogenesis Carcinog. Mutagen. 20:1-9, 2000.