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A new single nucleotide polymorphism was revealed using PCR—SSCP and sequencing methods within the bovine prolactin distal promoter region described as a functional enhancer. The A→G transition at position −1043 abolishes the recognition site for Hsp 92II restriction endonuclease, allowing for PCR—RFLP genotyping. The application of real-time PCR revealed that the prolactin gene expression level in the pituitary was higher in cattle with the AA genotype than in those with the GG genotype. EMSA analysis, however, showed increased nuclear protein binding to the sequence variant with G, suggesting a possible inhibition event, in which the transcription factors Pit1, Oct1, and YY1 could be involved.