The nucleotide sequence of a 3.6-kilobase pair DNA fragment containing the structural gene for yeast cytoplasmic lysyl-tRNA synthetase (KRS1) and its flanking regions was determined. The encoded protein of 67,881 kDa displays a cluster of 11 lysines within a 29-amino acid residue segment at its amino-terminal extremity. Evidence is presented that this segment is responsible for the affinity displayed by the native enzyme toward polyanionic carriers. The transcription initiation sites of the KRS1 gene were determined. Upstream from the TATA box, putative control elements corresponding to the concensus sequences for the RPG box and the general amino acid control system were identified. Evidence for transcriptional induction of the KRS1 gene via the general amino acid control system is presented.