This study examines whether the mimicking of selected properties of naturally occurring antisense RNAs in prokaryotes allows efficient inhibition of gene expression by in situ-expressed recombinant molecules in mammalian cells. Prokaryotic regulatory transcripts are expressed at high levels and have hairpin structures at their termini, features reminiscent of small nuclear RNAs (snRNAs) which are abundant and stable in the nucleus of all mammalian cells. A sequence complementary to fibrillin-1 (FBN1) mRNA, interrupted in its center by a hammerhead ribozyme, was substituted for the Sm protein binding site between the stem-loop structures of U1 snRNA. Expression of the chimeric antisense RNA resulted in dramatic inhibition of expression of fibrillin-1 message and protein in stably transfected cultured cells. The inhibitory effect was localized to the nucleus. The biological properties of U1 snRNA may provide a widely applicable vehicle for the in vivo delivery of antisense targeting sequences.