The mouse U1b SnRNA gene is expressed in only a limited range of cell types, whereas the U1a SnRNA gene is expressed in all cells. These two genes differ in the sequence of the octamer motif, which plays a critical role in SnRNA gene regulation. We show that the U1b octamer binds the octamer-binding protein Oct-1 with higher affinity than does the U1a octamer in both U1b-expressing and -non-expressing cell lines and tissues. Moreover, the U1b octamer can direct a higher level of gene expression than the U1a octamer when linked to a heterologous promoter and introduced into a non-U1b-expressing cell line. Hence the tissue-specific expression of the U1b gene is not determined by the failure of its octamer motif to bind Oct-1 or the weak affinity of this binding.