Splicing of the Drosophila P-element third intron (IVS3) is inhibited in somatic cells, restricting transposase expression to the germ line. Somatic inhibition of IVS3 splicing involves the assembly of a multiprotein complex on a regulatory sequence in the IVS3 5' exon. The P-element somatic inhibitor protein (PSI) is a component of this ribonucleoprotein complex and is required for inhibition of IVS3 splicing in vitro. The soma-specific expression pattern of PSI suggests that its low abundance in the germ line allows IVS3 splicing. We demonstrate that ectopic expression of PSI in the female germ line is sufficient to repress splicing of an IVS3 reporter transgene. We also show that IVS3 splicing is activated in somatic embryonic cells in the presence of an antisense PSI ribozyme. These results support the model that PSI is a tissue-specific regulator of IVS3 splicing in vivo.