The size of the 309 bp actin gene intron of the yeast Saccharomyces cerevisiae was enlarged by inserting DNA fragments of different lengths and sequence. Enlarging the intron above 551 bp, the largest known yeast intron, led to a decrease in splicing efficiency. The effect on transcript splicing was dependent on the length of the inserted fragments rather than their sequence. It was also observed that insertion of the actin gene intron into different regions of the normally unsplit yeast YP2 gene, significantly influenced the efficiency of splicing of the resulting transcripts. The splicing efficiency of splicing of with the increase of the distance between the mRNA cap site and the intervening sequence.