Publisher Summary This chapter reviews that the presence of the majority of the tRNAs inside an RNA tumor virus remains an enigma. The finding that specific tRNA molecules can serve as primers for DNA synthesis is of fundamental importance in the field of molecular biology. The transfer RNAs involved as primers are not restricted to a single tRNA species. The primer tRNA interacts at its 3' end with the template, and this interaction may involve as little as one-fourth of the tRNA molecule. Primer, but not nonprimer, tRNAs interact specifically with RNA-dependent DNA polymerases. On the basis of these properties, a number of techniques have been developed to identify primer tRNAs and to demonstrate their function. The chapter discusses that the insight and technology gained from studies of primer structure and function in RNA tumor virus systems should be helpful in the study of DNA replication in other systems. As more complex systems, including nuclei, are probed for the mechanisms by which DNA synthesis is initiated and controlled.