The Arabidopsis DNA ligase 1 gene (AtLIG1) is indispensable for cell viability. AtLIG1 expresses one major and two minor mRNA transcripts differing only in the length of the 5′ untranslated leader sequences preceding a common ORF. Control of AtLIG1 isoform production and intracellular targeting depends upon mechanisms controlling the choice of translation initiation site within the AtLIG1 ORF. Confocal laser scanning microscopy of green fluorescent protein-tagged AtLIG1 isoforms expressed in Arabidopsis revealed that translation of AtLIG1 mRNA transcripts from the first in-frame start codon produces an AtLIG1 isoform that is targeted exclusively to the mitochondria. Translation initiation from the second in-frame start codon produces an AtLIG1 isoform targeted only to the nucleus. There is no evidence for AtLIG1–GFP being targeted to chloroplasts. The mitochondrial AtLIG1 isoform possesses both an N-terminal mitochondrial-targeting signal and an internal bipartite nuclear localization signal (NLS) yet is targeted only to mitochondria, demonstrating a hierarchical dominance of the mitochondrial presequence over the NLS. The length of the 5′-UTR and more significantly the nucleotide context around alternative start codons in the AtLIG1 transcripts affect translation initiation to ensure a balanced synthesis of both nuclear and mitochondrial AtLIG1 isoforms, probably via a context-dependent leaky ribosome scanning mechanism.