Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.