We previously isolated a variant of the human U6 small nuclear RNA gene (87U6) and demonstrated that transcription of this gene is controlled by a novel internal promoter. It has now been shown that two blocks of sequence within the coding region are both necessary and sufficient to direct expression of 87U6 in transcription assays performed in vitro. In addition, 87U6 is expressed in vivo and can assemble into snRNP complexes. Specific primer extension assays on total RNA from HeLa cells shows that 87U6 RNA is present in these cells. Also, microinjection of plasmid encoded 87U6 genes into Xenopus laevis oocyte nuclei results in the expression of this variant RNA. Immunoprecipitation with anti-Sm antibodies suggests that 87U6 RNA assembles into a snRNP particle with U4 snRNA. Finally, the variant snRNA is capped with the U6 specific gamma-monomethyl phosphate cap when incubated in HeLa extracts. These data suggest that 87U6 RNA may function in the splicing process, in a manner similar to the wild-type U6 RNA. The recent observations of a minor class of mRNA introns that are spliced by a distinct collection of snRNP particles suggest an important role for variant snRNAs in the splicing of transcripts with alternative splice junctions.