Mitochondrial serine tRNA lacking D stem (anticodon AGY) was purified from bovine cardiac muscle in the mg order. The nucleotide sequence was identical to that reported previously, with t6A adjacent to the 3' end of anticodon (Arcari, P. and Brownlee, G.G. (1980) Nucleic Acids Res. 8 5207-5212). The procedures for large scale preparation and some properties of this tRNASer are reported.