The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and of 12 in vivo-generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D-loop, the intron and the extra arm affect the efficiency of splicing of the tRNA precursors while an anti-codon stem mutation may affect the accuracy of this process. Two mutations which occur in the intervening sequence of the sup9-e gene allow an alternate tRNA base pairing configuration. Transcription of the sup9-e gene and of the adjacent tRNAMet gene (located 7 bp downstream) is essentially abolished in vivo by a G----A19 mutation in the tRNASer gene, suggesting that tRNAMet may be derived solely via processing of the tRNASer-tRNAMet dimeric precursor.