The 5' flanking regions of the genes encoding human U1 and U2 snRNA each contain multiple copies of consensus pentanucleotide 5' GA/GGGC 3' which bind specifically to the SV40 T antigen. To examine the role of T antigen binding sites, the mutants deleted and substituted the pentanucleotide sequences from 5' flanking of U1 and U2 genes were constructed by oligonucleotide-directed DNA synthesis. A similar transcription level was observed between these mutants and wild type in cell-free transcription system. Both U1 and U2 snRNA synthesized in vitro initiate from upstream of the cap site identified in vivo. This imply that deletion and substitution of T antigen binding sites unaffect the transcriptional property of U1 and U2 genes in vitro. We have shown that deletion mutants virtually eliminated U1 and U2 snRNA synthesis in HeLa, 293 and oocytes cells, while substitution mutants fully restored transcription to wild type level. Thus, while the region located between -11 and -42 of human U1 or -58 and -90 of human U2 DNA is required for transcription in these cells, the wild type DNA sequence in this region is not a prerequisite for transcription. This suggests that this region is required for DNA spacing rather than nucleotide sequence.