We have analyzed the requirements for human U2 RNA transcription by injection of cloned U2/6 RNA genes into nuclei of Xenopus laevis oocytes. Two forms of human U2 RNAs accumulate, a major species corresponding to mature-sized U2 RNA and a minor species corresponding to a 3'-extended precursor. This RNA polymerase II transcription requires only 258 and 94 bp of 5'- and 3'-flanking region sequences, respectively. Efficient U2 RNA synthesis depends on a promoter element located between positions -258 and -198. This region contains a 12-bp direct repeat which strongly resembles a comparable upstream promoter element of the human U1 RNA genes. Sequences between -258 and -198 also confer on the U2 RNA template the ability to complete with co-injected U1 RNA templates for a snRNA gene-specific transcription factor(s). Transcription of U2 RNA is reduced off templates containing an active RNA polymerase III transcription unit, presumably because of relaxation or sequestration of the DNA. In vitro transcription of the U2 RNA gene, like that of the U1 RNA gene, is initiated upstream of the point corresponding to the 5' end of in vivo synthesized RNA.