The rice waxy (wx) locus has two functional alleles, Wxa and Wxb, which are defined by a large difference in the amount of the gene product, called Wx protein, that accumulates in mature seeds. To elucidate the molecular mechanism underlying this difference and to identify the base change causing the alteration of the regulation of the Wx gene during rice evolution, we determined the nucleotide sequences of the regulatory region of Wx alleles and analyzed their function in a transient assay system using rice protoplasts. All Wxa alleles from Oryza sativa Indica, O. rufipogon, and O. glaberrima have a normal sequence of GT at the 5' splice junction of the first intron, representing a high expression level of the Wx transcripts in the endosperm and a high beta-glucuronidase (GUS) activity in protoplasts. On the other hand, Wxb alleles from two strains of O. sativa Japonica have TT at the 5' splice junction, representing a low expression level of the mature transcripts and a low GUS activity. Northern blot analysis also indicated that a larger transcript, consisting of the unspliced first intron, is closely correlated with the function of the Wxb allele. These results suggest that a single base change at the 5' splice junction causes inefficient splicing and, as a result, reduces the level of mature transcript and the GUS activity in the Wxb allele. The Wxb allele in O. saativa Japonica may have been differentiated from the Wxa allele of O. rufipogon, its wild progenitor, by this mutation, and, therefore, a single base change that has altered the regulation of the Wx gene at the posttranscriptional level probably occurred during the domestication of rice.