A DNA fragment containing a mouse-thymidylate-synthase(TS) processed pseudogene was cloned and analyzed. Comparison with the sequences of the mouse TS-encoding gene (ts) and cDNA revealed that the pseudogene started at one of the normal 5' termini of TS mRNA, ended with a poly(A) tail, and was flanked by 16-nucleotide (nt) direct repeats. The region corresponding to the open reading frame was 97.3% identical to that of the cDNA. Two unusual features were observed. First, the poly(A) tail of the pseudogene was located 2 kb downstream from the normal location. Second, the final 10 nt of intron 5 were retained in the 'coding region' of the pseudogene. Therefore, it appears that the pseudogene was derived from a nonfunctional TS 'mRNA' that was aberrantly spliced and polyadenylated. Analysis of the sequence of intron 5 of the ts gene revealed the presence of an alternative 3' splice site 10 nt upstream from the normal splice site. S1-nuclease protection assays showed that about 10% of TS mRNA isolated from mouse cells was spliced at the alternative site.