We previously found that in sheep alpha alpha-, alpha alpha alpha-, and alpha alpha alpha alpha-globin gene haplotypes, in which individual genes encode distinct allelic variants, the gene expression determined at protein level progressively decreases from the 5' to the 3' end. In the present study, through direct DNA analysis, we definitively established the gene relative position in the cluster and we verified the occurrence of the gradient also at the mRNA level. We measured the relative abundance of the alpha 113His-mRNA in alpha alpha and alpha alpha alpha haplotypes, in which the gene coding for the alpha 113His chain occupies the second and the third position, respectively. The alpha 113His-mRNA levels were about 20% and 8% to 9%, respectively, which closely paralleled alpha-chain levels. We conclude that the expression gradient occurs also at the mRNA level and is most likely of transcriptional origin.