Eukaryotic splicing factors belonging to the SR family are essential splicing factors consisting of an N-terminal RNA-binding region and a C-terminal RS domain. They are believed to be involved in alternative splicing of numerous transcripts because their expression levels can influence splice site selection. We have characterized the structure and transcriptional regulation of the gene for the smallest member of the SR family, SRp20 (previously called X16). The mouse gene encoding SRp20, termed Srp20, consists of one alternative exon and six constitutive exons and was mapped to a 2-centimorgan interval on chromosome 17. When cells are transfected with SRp20 genomic DNA, both standard and alternatively spliced transcripts and corresponding proteins are produced. Interestingly, in starved (G 0) cells, the amount of SRp20 mRNA containing the alternative exon is large, whereas the amount of the standard SRp20 mRNA without the alternative exon is small. When starved cells are stimulated with serum, the alternative form is lost and the standard form is induced. These results suggest that splicing could be regulated during the cell cycle and that this could be, at least in part, due to regulated expression of SR proteins. Consistent with this, experiments with synchronized cells showed an induction of SRp20 transcripts in late G 1 or early S. We have also characterized the promoter of SRp20. It lies within a GC-rich CpG island and contains two consensus binding sites for E2F, a transcription factor thought to be involved in regulating the cell cycle. These motifs may be functional since reporter constructs with the SRp20 promoter can be stimulated by cotransfection with E2F expression plasmids.