From archaea to humans, a subset of transfer RNA (tRNA) genes possesses an intron that must be removed from transcribed pre-tRNAs to generate mature, functional tRNAs. Evolutionary conservation of tRNA intron sequences suggests that tRNA introns perform sequence-dependent cellular functions, which are presently unknown. Here, we demonstrate that free introns of tRNAs (fitRNAs) in Saccharomyces cerevisiae serve as small regulatory RNAs that inhibit mRNA levels via long (13-15 nt) statistically improbable stretches of (near) perfect complementarity to mRNA coding regions. The functions of fitRNAs are both constitutive and inducible because genomic deletion or inducible overexpression of tRNA