Many genes with important roles in development and disease contain exceptionally 
long introns, but special mechanisms for their expression have not been 
investigated. We present bioinformatic, phylogenetic, and experimental evidence 
in Drosophila for a mechanism that subdivides many large introns by recursive 
splicing at nonexonic elements and alternative exons. Recursive splice sites 
predicted with highly stringent criteria are found at much higher frequency than 
expected in the sense strands of introns >20 kb, but they are found only at the 
expected frequency on the antisense strands, and they are underrepresented 
within introns <10 kb. The predicted sites in long introns are highly conserved 
between Drosophila melanogaster and Drosophila pseudoobscura, despite extensive 
divergence of other sequences within the same introns. These patterns of 
enrichment and conservation indicate that recursive splice sites are 
advantageous in the context of long introns. Experimental analyses of in vivo 
processing intermediates and lariat products from four large introns in the 
unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these 
introns are removed by a series of recursive splicing steps using the predicted 
nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also 
confirmed that recursive splicing is the predominant processing pathway even 
with a shortened version of the intron. We discuss currently known and potential 
roles for recursive splicing.