Small poly(A)-containing RNA molecules which hybridize to the ubiquitous short repetitive sequence B2 and which are transcribed by RNA polymerase III have been identified in the cytoplasm of mouse cells. Here, we describe the structure of this small B2 RNA. A cDNA library was prepared from low-molecular-weight cytoplasmic poly(A)+RNA isolated from Ehrlich carcinoma cells and the clones which hybridized to B2 sequence were selected. The clones were sequenced and shown to contain B2 sequences followed by a poly(A) tract. The sequences of the cloned B2 RNAs different from each other by 3-10%, being similar in this respect to genomic B2 copies. Thus, B2 RNA is transcribed from many different B2 sequences in the genome. The 5'-ends of B2 RNA at least in most molecules coincide with the beginning of B2 genomic sequence. The poly(A) segments located at the 3'-end of small B2 RNA are the same size as in mRNA molecules, suggesting posttranscriptional formation. In some clones, additional sequences were detected between the 3'-end of B2 sequence and the poly(A) stretch. They seem to result from a lesion in the RNA polymerase III terminator in the corresponding B2 sequences. The possible significance of B2 sequences and small B2 RNA is discussed.