The steady-state levels of many mRNAs are determined in part by their turnover rates. Turnover rates, in turn, are usually controlled by proteins that bindcis-acting sequence elements in mRNAs. One class ofcis-acting instability determinants is composed of A+U-rich elements present in the 3′-UTRs of many labile mRNAs. Many A+U-rich elements are bound by the AUF1 family of RNA-binding proteins, which may target these mRNAs for rapid decay. cDNA cloning and immunoblot analyses suggest that the AUF1 family consists of at least four isoforms. Previous genomic cloning combined with FISH and Southern analyses of a panel of monochromosomal mouse/human or hamster/human somatic cell hybrids localized two AUF1 loci to human 4q21.1–q21.2 and Xq12 (B. Wagneret al.,1996,Genomics34: 219–222). In the present study AUF1 gene organization was examined. The results suggest that the four known AUF1 isoforms are generated by alternative pre-mRNA splicing of a transcript encoded by the chromosome 4 locus. Functionally, this creates isoforms with different RNA-binding affinities and specificities. Thus, alternative pre-mRNA splicing may serve to create functional versatility within the AUF1 family of proteins.