As the genomic revolution continues to evolve, there is an increasing demand for efficient and reliable tools for functional characterization of individual gene products. Antisense oligonucleotide-mediated knockdown has been used successfully as a functional genomics tool in animal models of pain and analgesia yet skepticism regarding the validity and utility of antisense technology remains. Contributing to this uncertainty are the lack of systematic studies exploring antisense oligonucleotide use in vivo and the many technical and methodological challenges intrinsic to the method. This article reviews the contributions of antisense oligonucleotide-based studies to the field of pain and analgesia and the general principles of antisense technology. A special emphasis is placed on technical issues surrounding the successful application of antisense oligonucleotides in vivo, including sequence selection, antisense oligonucleotide chemistry, DNA controls, route of administration, uptake, dose-dependence, time-course and adequate evaluation of knockdown.