The status of the p53 gene in SEWA-AS12-ADH (S-ADH) cells, a subline of the mouse sarcoma cell line SEWA, was examined. Immunoprecipitation with wild-type (wt) or mutant P53-specific monoclonal antibodies (mAb) showed that both wt and mutant P53 were produced. Sequence analysis of the p53 cDNA and genomic DNA revealed a single nucleotide (nt) substitution at a splice donor site at the beginning of intron 7. As a result of this mutation, an alternative splice site 15 nt further 3' in intron 7 is used. The P53 protein translated from this aberrantly spliced mRNA carries an Arg258-->Ser substitution, followed by an insertion of 5 extra amino acids. This is the first example of a splice-site mutation in the mouse p53 gene.