Abstract: In the alternative splicing, intron retention, of histamine H₃ receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. Theretained introns have common 5′ splice site and different 3′ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptorsand investigated the effects of 5′ and 3′ splice sites on intron retention in HeLa cells. Mutating weak 5′ and 3′ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouseminigenes. The effect of splice site strength was context-dependent and much more significant for the 3′ splice site of the longer alternative intron than for the 3′ splice site of the shorter alternative intron and the common 5′ splice sites; it was also more significant in the ratminigene than in the mouse minigene. Mutating the 3′ splice site of the longer alternative intron resulted in almost complete splicing ofthe intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.