In mouse, U3B small nuclear RNA is encoded by a small family of four functional genes. Three of these genes have recently been identified, with two of them being located 5 kb apart, in the same orientation [Mazan and Bachellerie, J. Biol. Chem. 263 (1988) 19461-19467]. By analyzing a cosmid library of mouse genomic DNA we have isolated the fourth gene, U3B.4, which is located 12 kb downstream from the previously reported pair of linked genes, U3B.1 and U3B.2, in the same orientation. Its sequence analysis demonstrates that four mouse U3B genes have undergone a very efficient concerted evolution, with a perfect sequence identity not only over the entire RNA coding region but also over 254 bp of 5' flanking DNA which contain essential transcription signals. Moreover, in the regions which immediately flank the domain of perfect sequence identity, a patchwork of sequence similarities among the four genes is apparent, with a series of striking reversals in relationships between adjacent localized areas. This suggests that gene conversions have played a major role in the concerted evolution of the U3B gene family. A superimposition of conversion events, which probably occurred at distinct stages of rodent evolutions can be inferred. This has involved not only the three clustered copies, but also the remaining gene, U3B.3, the location of which relative to the cluster remains unknown. B1 repetitive elements are found at the boundaries of conversion domains, suggesting their possible role in control of the sequence homogenization process.