Quantification of β-lactoglobulin A and B in the milk of heterozygous animals has revealed a differential content of these two variants. Nucleotide sequence analysis of the first 733 bp of the bovine β-lactoglobulin promoter has indicated the presence of ten polymorphic sites, nine of them being allele A and B specific mutations. To study the differential expression of these alleles, constructs containing 753 bp long fragments of the bovine β-lactoglobulin A and B associated promoters were used in transient transfection experiments in HC11 cells. A differential transcription activity directed by the A and B specific promoters was consistently observed. The relative expression levels were 57% for β-lactoglobulin A and 43% for β-lactoglobulin B promoters. An allele-specific mutation has been reported to have a differential binding affinity to the activator protein-2 between the β-lactoglobulin A and B promoters. However, experiments in HC11 cells where the activator protein-2 binding sites were mutated in both β-lactoglobulin A and B promoters showed no major differences in activity between the mutated and native promoters.