We previously described a family of five RNA-binding proteins: CUG-binding protein, embryonic lethal abnormal vision-type RNA-binding protein 3, and the CUGbinding protein and embryonic lethal abnormal visiontype RNA-binding protein 3-like factors (CELFs) 3, 4, and 5. We demonstrated that all five of these proteins specifically activate exon inclusion of cardiac troponin T minigenes in vivo via muscle-specific splicing enhancer (MSE) sequences. We also predicted that a sixth family member, CELF6, was located on chromosome 15. Here, we describe the isolation and characterization of CELF6. Like the previously described CELF proteins, CELF6 shares a domain structure containing three RNA-binding domains and a divergent domain of unknown function. CELF6 is strongly expressed in kidney, brain, and testis and is expressed at very low levels in most other tissues. In the brain, expression is widespread and maintained from the fetus to the adult. CELF6 activates exon inclusion of a cardiac troponin T minigene in transient transfection assays in an MSE-dependent manner and can activate inclusion via multiple copies of a single element, MSE2. These results place CELF6 in a functional subfamily of CELF proteins that includes CELFs 3, 4, and 5. CELF6 also promotes skipping of exon 11 of insulin receptor, a known target of CELF activity that is expressed in kidney.Alternative splicing allows the production of multiple mRNA species from a single gene, which often give rise to functionally distinct protein isoforms. Regulation of alternative splicing not only produces multiple mRNAs but can also modulate the levels of these different isoforms in a tissue-or developmental stage-specific manner to meet the functional needs of the cell. Numerous examples of regulated alternative splicing have been found, but few regulatory factors that control cell-specific alternative splicing have been identified. In the best-characterized vertebrate experimental systems, alternative splicing regulation is the result of dynamic antagonism between transacting factors binding to positive or negative elements in the pre-mRNA and promoting or repressing the use of alternative splice sites. Some of these elements bind cell-specific splicing factors. For example, neuron-specific inclusion of alternative exons in several pre-mRNAs is mediated by binding of the activator Nova-1, which is expressed only in neurons. This neuron-specific activity is antagonized by binding of the ubiquitously expressed repressor polypyrimidine tract-binding protein (PTB) in non-neuronal cells (1).The muscle-specific alternative exon 5 of cardiac troponin T (cTNT) 1 is also regulated by antagonistic activities. Musclespecific splicing enhancers (MSEs) in cTNT pre-mRNAs regulate inclusion of exon 5 in striated muscle by binding to activators and repressors of splicing. Negative elements within MSEs upstream and downstream of exon 5 repress exon inclusion in non-muscle cells by binding PTB (2).