A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments. Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA). Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site. Of these, only one, SC99, appeared to be a specificity mutant. Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant. The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition. Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant. These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.