Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis-and trans-regulatory signals. The extent of exon 2 inclusion is stage-and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.The vast majority of metazoan genes contain short, information-encoding exons interspersed by relatively long stretches of noncoding introns. The processing of this information to yield a translatable message, including the splicing of the pre-mRNA, is subject to regulation at virtually every definable step. One such step, including or excluding a particular exon-alternative splicing-provides a way to alter the functional activity of a protein in the absence of gene duplication (1, 2). Discreet functional domains may be added or subtracted depending on signals elaborated by the particular needs of the cell. The study of alternative splicing further provides an experimental tool for understanding how the participating ribonucleoprotein complexes target intron-exon boundaries (3).Vertebrate exons are, on average, less than 300 nucleotides, whereas many introns are thousands of nucleotides in length (4). Exons below an average size of 50 nucleotides have been shown to be inherently difficult to recognize by the splicing machinery (4, 5). One explanation for this "masking" of small exons, originally set forth by Berget (5), is that the initiation of the splicing reaction is exon-centered. There is much evidence to support this model, termed exon definition, in which ribonucleoprotein initiation complexes recognize intron-exon boundaries and bridge across the exon. As exon size decreases below 50 nucleotides, these complexes are prevented from forming a productive interaction with the 3Ј-and 5Ј-splice recognition sites on the pre-mRNA or with each other, through steric hindrance. Sequences that function either to facilitate (enhancers) or to inhibit (repressors) the recognition of alternatively spliced exons have been found in both introns (6 -9) and exons (9 -11).Adenosine monophosphate deaminase (AMPD) 1...