A DNA-dependent in vitro transcription system for the human U1 small nuclear RNA (snRNA) promoter has been developed. This in vitro transcription system uses extracts of tissue culture cells to drive transcription of an RNA polymerase II-transcribed snRNA gene. A U1 promoter (-393 to +192) template was constructed in which the sequences from +10 to +171 were replaced with a 179-bp sequence from a G-less cassette. This DNA template thus retained all of the known U1 promoter elements, including the U1 3'-end box (positions +175 to +191), which is responsible for snRNA 3'-end formation. HeLa cell nuclear extracts were shown to drive specific transcription of this promoter by RNA polymerase II. This transcription system has many of the properties observed for wild-type snRNA promoters in vivo. Transcription was shown to initiate at +1 (and -2) relative to the U1 promoter and to efficiently (greater than 90%) form a 3' end corresponding to the 3' end found in the primary transcript of U1 in vivo. The transcription signal is responsive to either deletion or replacement of the U1 distal sequence (enhancer-like) and proximal sequence (TATA-like) elements, as well as the 3'-end box. Additionally, the signal was shown by depletion/repletion experiments to be responsive to a protein called PSE1 (related to Ku), which has recently been shown to specifically bind sequences in the U1 promoter. This in vitro snRNA transcription system should facilitate the biochemical analysis of the human U1 snRNA promoter and lead to a better understanding of the differences between snRNA and mRNA promoters.