The α-globin major regulatory element (αMRE) positioned far upstream of the gene cluster is essential for the proper expression of the α-globin genes. Analysis of the human and mouse α-globin Upstream Flanking Regions (αUFR) has identified three nonglobin genes in the order Dist1-MPG-Prox1-α-globin. Further characterization of the whole region indicates that the αMRE and several other erythroid DNase HSSs are associated with the transcription unit of the Prox1 gene. In this paper we describe the characterization and localization of the mouse Prox1 cDNA and compare it with its human homolog, the -14 gene, and another human cDNA sequence named hProx1. Our results show a strong conservation between the -14 gene and the mouse Prox1 gene with the exception of the first exon of the mProx1 gene. This exon is absent in the -14 cDNA but is present and conserved in the human Prox1 cDNA, indicating that the human -14/hProx1 gene is alternatively spliced or transcribed. The mProx1 gene encodes a predicted protein of 491 amino acids (aa) whose function is not known. In the 5′UTR of this gene, a 35-bp repeat (VNTR) is positioned, which is highly polymorphic among laboratory inbred mice (Mus domesticus). Our results strongly suggest that the mProx1 VNTR arose during the divergence of M. spretus and M. domesticus. Besides its use in evolutionary studies and positional cloning, the mProx1 VNTR might be invaluable for monitoring the expression of a transgenic mProx1 gene. The cloning of the mProx1 gene will be helpful to analyze its possible role on α-globin as well on MPG expression in the mouse.