A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.