The structure of rabbit liver aspartate tRNA 2 was derived by two postlabelling techniques involving labelling at 3′ or 5′ end followed by controlled hydrolysis with base-specific nucleases and product characterization by gel electrophoresis: ▪ This tRNA of 76 residues contains 8 modified nucleotides (1-MeAdo, rThd, H 2Urd, Quo, two each of 5-MeCyd and ψrd). Although the proposed sequence resembles that of a recently described “major” isoacceptor of aspartate tRNA from rat liver, it differs in 13 nucleotides and contains an additional residue in the variable loop. Our tRNA sequence shows 65 per cent homology with an isoacceptor from the yeast, but only 55 per cent with the isoacceptor from Escherichia coli, and has very little resemblance to the aspartate isoacceptors from normal or tumor mitochondria.