The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been studied by microinjection into Xenopus oocytes. At least two distinct regions in the 5'-flanking DNA contribute to U4B RNA gene expression. The proximal regulatory element, which is inactivated by a 5'-flanking DNA deletion to position -38, provides a basal level of U4B RNA synthesis. The distal regulatory region, centered near position -200, acts as a transcriptional enhancer. It provides a 4-5 fold stimulation of U4B RNA gene expression above the basal level, and, like mRNA enhancers, is composed of multiple functional motifs. One of these, the octamer sequence ATGCAAAG, has previously been recognized as an important element of U1 and U2 snRNA gene enhancers, as well as being involved in the expression of a number of mRNA genes. However, the octamer sequence is not sufficient for U4B enhancer activity. An additional element, an "Sph motif," is located 12 base pairs downstream of the octamer and is an essential component of the U4B enhancer. Transcriptional competition studies indicate that the U4B and U1 snRNA genes utilize a common set of transcription factors.