We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.