We constructed rabbit beta-globin genes with deletions in the large intron, extending from the midpoint toward the 5' or 3' splice sites. Analysis of transcripts in transformed HeLa cells showed that six 5' proximal intron nucleotides allowed normal splicing. Correct splicing at the 3' splice site required 12 or more 3' proximal intron nucleotides; optimal efficiency required 24 nucleotides. Remarkably, a mini-intron comprising six 5' and 24 3' intron nucleotides gave no correctly spliced transcripts; extending the miniintron with polyoma or pBR322 fragments to 80 or more nucleotides restored normal splicing. Thus other than in yeast nuclear genes, no specific internal intron sequences appear to be needed but a minimal intron length is important.