spl1-1 was originally identified as a spontaneous mutation genetically interacting with sep1-1 and cdc4-8 in producing multinucleate syncytia. This study shows that it is allelic with the proline-tRNA(CGG) gene SPATRNAPRO.02. Its nucleotide sequence contains a C-->T substitution in the region corresponding to the B-box of the putative intragenic promoter and the TpsiC loop of the mature tRNA. The substitution drastically reduces the transcription efficiency of the gene and pleiotropically affects numerous cellular processes. spl1-1 cells are temperature sensitive, osmosensitive, bend at higher temperatures, have extended G2 phase and are defective in cell separation (septum cleavage). The proline-tRNA(TGG) gene SPATRNAPRO.01 can partially suppress the spl1-1 mutation when introduced into the cells on a multicopy plasmid. The effect of a mutation in a tRNA gene on cell separation brings a new element into the complexity of the regulation of cell division and its co-ordination with other cellular processes in Schizosaccharomyces pombe.